Biology Protocols

Preparation of Plasmid DNA by Large-scale Boiling Lysis

2008-04-24T13:18:00.001-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is isolated from large-scale (500 ml) bacterialcultures by treatment with Triton X-100 and lysozyme, followedby heating. This method is not recommended for preparing plasmidDNA from strains of E. coli that express endonuclease A (endA⁺strains).

MATERIALS

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Ethanol

Optional, please see Step 5.

Isopropanol

Lysozyme(10 mg/ml)

Prepare the solution fresh in Tris-Cl (pH 8.0).

Restriction endonucleases

Please see Step 4.

Rich medium

STE

STET

TE (pH 8.0)

METHOD

1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth)containing the appropriate antibiotic either with a single colonyof transformed bacteria or with 0.1-1.0 ml of a small-scaleliquid culture grown from a single colony.

2. Incubate theculture at the appropriate temperature withvigorous shaking(250 cycles/ minute in a rotary shaker) untilthe bacteria reachthe late log phase of growth (i.e., an OD₆₀₀of approx. 0.6).

3. Inoculate 500 ml of LB, YT, or Terrific Broth (prewarmedto 37°C) containing the appropriate antibiotic in a 2-literflask with 25 ml of the late-log-phase culture. Incubate theculture for 2.5 hours at 37°C with vigorous shaking (250cycles/minute on a rotary shaker).

4. Add 2.5 ml of 34 mg/mlchloramphenicol. The final concentrationof chloramphenicolin the culture should be 170 µg/ml.Incubate the culturefor a further 12-16 hours at 37°C withvigorous shaking(250 cycles/minute on a rotary shaker).

5. Remove an aliquot(1-2 ml) of the bacterial culture to afresh microcentrifugetube and store at 4°C. Harvest theremainder of the bacterialcells from the 500-ml culture bycentrifugation at 2700g (4100rpm in a Sorvall GSA rotor) for15 minutes at 4°C. Discardthe supernatant. Stand the opencentrifuge bottle in an invertedposition to allow all of thesupernatant to drain away.

6.Resuspend the bacterial pellet in 200 ml of ice-cold STE.Collectthe bacterial cells by centrifugation as described inStep 5.Store the pellet of bacteria in the centrifuge bottleat -20°C.

7. Prepare plasmid DNA from the 1-2-ml aliquot of bacteriasetaside in Step 5 by the minipreparation protocol (eitherPreparation of Plasmid DNA by Alkaline Lysis with SDS: MinipreparationorRemoval of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation through NaCl).Analyze the minipreparation plasmid DNA by digestion with theappropriate restriction enzyme(s) to ensure that the correctplasmid has been propagated in the large-scale culture.

8.Allow the frozen bacterial cell pellet from Step 6 to thawfor5-10 minutes at room temperature. Resuspend the pellet in10ml of ice-cold STET. Transfer the suspension to a 50-ml Erlenmeyerflask.

9. Add 1 ml of a freshly prepared solution of 10 mg/mllysozyme.

10. Use a clamp to hold the Erlenmeyer flask overthe open flameof a Bunsen burner until the liquid just startsto boil. Shakethe flask constantly during the heating procedure.

11. Immediately immerse the bottom half of the flask in alarge(2-liter) beaker of boiling water. Hold the flask in theboilingwater for exactly 40 seconds.

12. Cool the flask inice-cold water for 5 minutes.

13. Transfer the viscous contentsof the flask to an ultracentrifugetube (Beckman SW41 or itsequivalent). Centrifuge the lysateat 150,000g (30,000 rpm ina Beckman SW41Ti rotor) for 30 minutesat 4°C.

14. Transferas much of the supernatant as possible to a newtube. Discardthe viscous liquid remaining in the centrifugetube.

15. (Optional)If the supernatant contains visible strings ofgenomic chromatinor flocculent precipitate of proteins, filterit through 4-plygauze before proceeding.

16. Measure the volume of the supernatant.Transfer the supernatant,together with 0.6 volume of isopropanol,to a fresh centrifugetube(s). Store the tube(s) for 10 minutesat room temperature,after mixing the contents well.

17. Recoverthe precipitated nucleic acids by centrifugationat 12,000g(10,000 rpm in a Sorvall SS-34 rotor) for 15 minutesat roomtemperature.

Salt may precipitate if centrifugation is carriedout at 4°C.

18. Decant the supernatant carefully, andinvert the open tube(s)on a paper towel to allow the last dropsof supernatant to drainaway. Rinse the pellet and the wallsof the tube(s) with 70%ethanol at room temperature. Drain offthe ethanol, and usea Pasteur pipette attached to a vacuumline to remove any beadsof liquid that adhere to the wallsof the tube(s). Place theinverted, open tube(s) on a pad ofpaper towels for a few minutesat room temperature. The pelletshould still be damp.

19. Dissolve the pellet of nucleic acidin 3 ml of TE (pH 8.0).

20. Purify the crude plasmid DNA eitherby chromatography oncommercial resins (Purification of Plasmid DNA by Chromatography),precipitation with polyethylene glycol (Purification of Plasmid DNA by Precipitation with Polyethylene Glycol),or equilibrium centrifugation in CsCl-ethidium bromide gradients(Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous GradientsandPurification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients).

21. Check the structure of the plasmid by restriction enzymedigestion followed by gel electrophoresis.

REFERENCES

1. Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.

Caution

Lysozyme

Lysozyme is caustic to mucous membranes. Wear appropriate glovesand safety glasses.

Caution

Radioactive substances

Radioactive substances: When planning an experiment that involvesthe use of radioactivity, consider the physico-chemical propertiesof the isotope (half-life, emission type, and energy), the chemicalform of the radioactivity, its radioactive concentration (specificactivity), total amount, and its chemical concentration. Orderand use only as much as needed. Always wear appropriate gloves,lab coat, and safety goggles when handling radioactive material.X-rays and gamma rays are electromagnetic waves of very shortwavelengths either generated by technical devices or emittedby radioactive materials. They might be emitted isotropicallyfrom the source or may be focused into a beam. Their potentialdangers depend on the time period of exposure, the intensityexperienced, and the wavelengths used. Be aware that appropriateshielding is usually made of lead or other similar material.The thickness of the shielding is determined by the energy(s)of the X-rays or gamma rays. Consult the local safety officefor further guidance in the appropriate use and disposal ofradioactive materials. Always monitor thoroughly after usingradioisotopes. A convenient calculator to perform routine radioactivitycalculations can be found at:http://www.graphpad.com/calculators/radcalc.cfm.

Recipe

Lysozyme

(10 mg/ml) Dissolve solid lysozyme at a concentration of 10mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before use. Makesure that the pH of the Tris solution is 8.0 before dissolvingthe protein. Lysozyme will not work efficiently if the pH ofthe solution is less than 8.0.

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

STE

10 mM Tris-Cl (pH 8.0)

0.1 M NaCl

1 mM EDTA (pH 8.0)

Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm²)on liquid cycle. Store the sterile solution at 4°C.

Recipe

STET

0.1 M NaCl

5% (v/v) Triton X-100

10 mM Tris-Cl (pH 8.0)

1 mM EDTA (pH 8.0)

Make sure that the pH of STET is 8.0 after all ingredients areadded. There is no need to sterilize STET before use.

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Preparation of Plasmid DNA by Large-scale Boiling Lysis

2008-04-24T13:18:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

MATERIALS

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Ethanol

Optional, please see Step 5.

Isopropanol

Lysozyme(10 mg/ml)

Prepare the solution fresh in Tris-Cl (pH 8.0).

Restriction endonucleases

Please see Step 4.

Rich medium

STE

STET

TE (pH 8.0)

METHOD

6.Resuspend the bacterial pellet in 200 ml of ice-cold STE.Collectthe bacterial cells by centrifugation as described inStep 5.Store the pellet of bacteria in the centrifuge bottleat -20°C.

8.Allow the frozen bacterial cell pellet from Step 6 to thawfor5-10 minutes at room temperature. Resuspend the pellet in10ml of ice-cold STET. Transfer the suspension to a 50-ml Erlenmeyerflask.

9. Add 1 ml of a freshly prepared solution of 10 mg/mllysozyme.

10. Use a clamp to hold the Erlenmeyer flask overthe open flameof a Bunsen burner until the liquid just startsto boil. Shakethe flask constantly during the heating procedure.

11. Immediately immerse the bottom half of the flask in alarge(2-liter) beaker of boiling water. Hold the flask in theboilingwater for exactly 40 seconds.

12. Cool the flask inice-cold water for 5 minutes.

13. Transfer the viscous contentsof the flask to an ultracentrifugetube (Beckman SW41 or itsequivalent). Centrifuge the lysateat 150,000g (30,000 rpm ina Beckman SW41Ti rotor) for 30 minutesat 4°C.

14. Transferas much of the supernatant as possible to a newtube. Discardthe viscous liquid remaining in the centrifugetube.

15. (Optional)If the supernatant contains visible strings ofgenomic chromatinor flocculent precipitate of proteins, filterit through 4-plygauze before proceeding.

17. Recoverthe precipitated nucleic acids by centrifugationat 12,000g(10,000 rpm in a Sorvall SS-34 rotor) for 15 minutesat roomtemperature.

Salt may precipitate if centrifugation is carriedout at 4°C.

19. Dissolve the pellet of nucleic acidin 3 ml of TE (pH 8.0).

21. Check the structure of the plasmid by restriction enzymedigestion followed by gel electrophoresis.

REFERENCES

1. Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.

Caution

Lysozyme

Lysozyme is caustic to mucous membranes. Wear appropriate glovesand safety glasses.

Caution

Radioactive substances

Recipe

Lysozyme

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

STE

10 mM Tris-Cl (pH 8.0)

0.1 M NaCl

1 mM EDTA (pH 8.0)

Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm²)on liquid cycle. Store the sterile solution at 4°C.

Recipe

STET

0.1 M NaCl

5% (v/v) Triton X-100

10 mM Tris-Cl (pH 8.0)

1 mM EDTA (pH 8.0)

Make sure that the pH of STET is 8.0 after all ingredients areadded. There is no need to sterilize STET before use.

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Purification of Plasmid DNA by Precipitation with Polyethylene Glycol

2008-04-24T13:16:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Crude preparations of plasmid DNA are first treated with lithiumchloride and RNase (to remove RNA). The plasmid DNA is thenprecipitated in a solution containing polyethylene glycol andMgCl₂.

MATERIALS

Chloroform

Crude plasmid preparation

Use material from either Step 17 of Preparation of Plasmid DNA by Alkaline Lysis with SDS: MaxipreparationorStep 19 of Preparation of Plasmid DNA by Large-scale Boiling Lysis.

Ethanol

Optional, please see Step 5.

Isopropanol

LiCl(5 M)

PEG-MgCl₂ solution

Phenol:chloroform (1:1,v/v)

Sodiumacetate (3 M, pH 5.2)

TE (pH 8.0)

TE (pH 8.0) containing 20µg/ml RNase A

METHOD

1. Transfer 3 ml of the crude large-scale plasmid preparationto a 15-ml Corex tube and chill the solution to 0°C in anice bath.

2. Add 3 ml of an ice-cold solution of 5 M LiClto the crudeplasmid preparation, mix well, and centrifuge thesolution at12,000g (10,000 rpm in a Sorvall SS-34 rotor) for10 minutesat 4°C.

3. Transfer the supernatant to a fresh30-ml Corex tube. Addan equal volume of isopropanol. Mix well.Recover the precipitatednucleic acids by centrifugation at12,000g (10,000 rpm in aSorvall SS-34 rotor) for 10 minutesat room temperature.

4. Decant the supernatant carefully,and invert the open tubeto allow the last drops of supernatantto drain away. Rinsethe pellet and the walls of the tube with70% ethanol at roomtemperature. Carefully discard the bulkof the ethanol, andthen use a vacuum aspirator to remove anybeads of liquid thatadhere to the walls of the tube. Placethe inverted, open tubeon a pad of paper towels for a few minutes.The pellet shouldstill be damp.

5. Dissolve the damp pelletof nucleic acid in 500 µlof TE (pH 8.0) containing RNaseA. Transfer the solution toa microcentrifuge tube and storeit for 30 minutes at room temperature.

6. Extract the plasmid-RNasemixture once with phenol:chloroformand once with chloroform.

7. Recover the DNA by standard ethanol precipitation.

8.Dissolve the pellet of plasmid DNA in 1 ml of sterile H₂O,andthen add 0.5 ml of PEG-MgCl₂ solution.

9. Store the solutionfor 10 minutes at room temperature, andthen collect the precipitatedplasmid DNA by centrifugationat maximum speed for 20 minutesat room temperature in a microcentrifuge.

10. Remove tracesof PEG by resuspending the pellet of nucleicacid in 0.5 mlof 70% ethanol. Collect the nucleic acid by centrifugationatmaximum speed for 5 minutes in a microcentrifuge.

11. Removethe ethanol by aspiration and repeat Step 10. Followingthesecond rinse, store the open tube on the bench for 10-20minutesto allow the ethanol to evaporate.

12. Dissolve the damp pelletin 500 µl of TE (pH 8.0).Measure the OD₂₆₀ of a 1:100dilution in TE (pH 8.0) of thesolution, and calculate the concentrationof the plasmid DNAassuming that 1 OD₂₆₀ = 50 µg of plasmidDNA/ml.

13. Store the DNA in aliquots at -20°C.

REFERENCES

1. Nicoletti, V.G. and Condorelli, D.F. 1993. Optimized PEG-method for rapid plasmid DNA purification: High yield from "midiprep." BioTechniques 14: 532–534.

2. Nicoletti, V.G. and Condorelli, D.F. 1993. Optimized PEG-method for rapid plasmid DNA purification: High yield from "midiprep." BioTechniques 14: 536–536.

3. Lis, J.T. 1980. Fractionation of DNA-fragments by polyethylene glycol induced precipitation. Methods Enzymol. 65: 347–353.

Caution

Chloroform

Chloroform CHCl₃ is irritating to the skin, eyes, mucous membranes,and respiratory tract. It is a carcinogen and may damage theliver and kidneys. It is also volatile. Avoid breathing thevapors. Wear appropriate gloves and safety glasses. Always usein a chemical fume hood.

Caution

LiCl

LiCl, see Lithium chloride

Caution

Phenol:chloroform

Phenol is extremely toxic, highly corrosive, and can cause severeburns. It may be harmful by inhalation, ingestion, or skin absorption.Wear appropriate gloves, goggles, and protective clothing. Alwaysuse in a chemical fume hood. Rinse any areas of skin that comein contact with phenol with a large volume of water and washwith soap and water; do not use ethanol!

Chloroform (CHCl₃) is irritating to the skin, eyes, mucous membranes,and respiratory tract. It is a carcinogen and may damage theliver and kidneys. It is also volatile. Avoid breathing thevapors. Wear appropriate gloves and safety glasses. Always usein a chemical fume hood.

Caution

Sodium acetate (NaOAc)

Sodium acetate (NaOAc), see Acetic acid

Recipe

LiCl

To prepare 10 M lithium chloride: Dissolve 42.4 g of LiCl ina final volume of 90 ml of H₂O. Adjust the volume of the solutionto 100 ml with H₂O. Sterilize the solution by passing it througha 0.22-µm filter, or by autoclaving for 15 minutes at15 psi (1.05 kg/cm 2) on liquid cycle. Store the solution at4°C.

Recipe

PEG-MgCl₂ Solution

40%(w/v) polyethylene glycol (PEG 8000)

30mM MgCl₂

Dissolve 40 g of PEG 8000 in a final volume of 100 ml of 30mM MgCl₂. Sterilize the solution by passing it through a 0.22-µmfilter, and store it at room temperature.

Recipe

Sodium acetate

To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate•3H₂Oin 800 mL of H₂O. Adjust the pH to 5.2 with glacial acetic acidor to 7.0 with dilute acetic acid. Adjust the volume to 1 Lwith H₂O. Dispense into aliquots and sterilize by autoclaving.

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Preparation of Plasmid DNA by Lysis with SDS

2008-04-24T13:14:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Large (>15 kb), closed circular plasmids are prepared (albeitinefficiently and in small yield) by lysing bacteria with SDS.

MATERIALS

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Chloroform

Optional, please see Step 7.

EDTA (0.5 M, pH 8.0)

Ethanol

Optional, please see Step 5.

Lysozyme(10 mg/ml)

Prepare the solution fresh in Tris-Cl (pH 8.0).

NaCl (5 M)

Store the solution of NaCl at 4°C.

Phenol:chloroform (1:1,v/v)

Restriction endonucleases

Please see Step 4.

Rich medium

SDS(10% w/v)

STE, ice cold

TE (pH 8.0)

Tris-sucrose

METHOD

1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth)containing the appropriate antibiotic with a single transformedbacterial colony or with 0.1-1.0 ml of a late-log-phase culturegrown from a single transformed colony.

2. Incubate the culturewith vigorous shaking until the bacteriaenters the late logphase of growth (i.e., an OD₆₀₀ of approx.0.6).

3. Inoculate500 ml of LB, YT, or Terrific Broth (prewarmedto 37°C)containing the appropriate antibiotic in a 2-literflask with25 ml of the late-log-phase culture. Incubate theculture forapprox. 2.5 hours at 37°C with vigorous shaking(250 cycles/minuteon a rotary shaker).

4. For relaxed plasmids with low or moderatecopy numbers, add2.5 ml of 34 mg/ml chloramphenicol. The finalconcentrationof chloramphenicol in the culture should be 170µg/ml.For high-copy-number plasmids, do not add chloramphenicol.

5. Incubate the culture for a further 12-16 hours at 37°Cwith vigorous shaking (250 cycles/minute on a rotary shaker)

6. Remove an aliquot (1-2 ml) of the bacterial culture toafresh microcentrifuge tube and store it at 4°C. Harvestthe remainder of the bacterial cells from the 500-ml cultureby centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor)for 15 minutes at 4°C. Discard the supernatant. Stand theopen centrifuge bottle in an inverted position.

7. Resuspendthe bacterial pellet in 200 ml of ice-cold STE.Collect thebacterial cells by centrifugation as described inStep 6. Storethe pellet of bacteria in the centrifuge bottleat -20°C.

8. Use one of the methods described in Preparation of Plasmid DNA by Alkaline Lysis with SDS: MinipreparationorPreparation of Plasmid DNA by Small-scale Boiling Lysisto prepareplasmid DNA from the 1-2-ml aliquot of bacterial culture setaside in Step 6. Analyze the minipreparation plasmid DNA bydigestion with the appropriate restriction enzyme(s) and agarosegel electrophoresis to ensure that the correct plasmid has beenpropagated in the large-scale culture.

9. Allow the frozenbacterial cell pellet from Step 7 to thawat room temperaturefor 5-10 minutes. Resuspend the pellet in10 ml of ice-coldTris-sucrose solution. Transfer the suspensionto a 30-ml plasticscrew-cap tube.

10. Add 2 ml of a freshly prepared lysozymesolution (10 mg/ml)followed by 8 ml of 0.25 M EDTA (pH 8.0).

11. Mix the suspension by gently inverting the tube severaltimes. Store the tube on ice for 10 minutes.

12. Add 4 mlof 10% SDS. Immediately mix the contents of thetube with aglass rod so as to disperse the solution of SDSevenly throughoutthe bacterial suspension. Be as gentle aspossible to minimizeshearing of the liberated chromosomal DNA.

13. As soon asmixing is completed, add 6 ml of 5 M NaCl (finalconcentration= 1 M). Use a glass rod to mix the contents ofthe tube gentlybut thoroughly. Place the tube on ice for atleast 1 hour.

14.Remove high-molecular-weight DNA and bacterial debris bycentrifugationat 71,000g (30,000 rpm in a Beckman Type 50 rotor)for 30 minutesat 4°C. Carefully transfer the supernatantto a 50-ml disposableplastic centrifuge tube. Discard the pellet.

15. Extract thesupernatant once with phenol:chloroform andonce with chloroform.

16. Transfer the aqueous phase to a 250-ml centrifuge bottle.Add 2 volumes (approx. 60 ml) of ethanol at room temperature.Mix the solution well. Store the solution for 1-2 hours at roomtemperature.

17. Recover the nucleic acids by centrifugationat 5000g (5500rpm in a Sorvall GSA rotor or 5100 rpm in a SorvallHS4 swing-outrotor) for 20 minutes at 4°C.

18. Discardthe supernatant. Wash the pellet and sides of thecentrifugetube with 70% ethanol at room temperature and thencentrifugeas in Step 17.

19. Discard as much of the ethanol as possible,and then invertthe centrifuge bottle on a pad of paper towelsto allow thelast of the ethanol to drain away. Use a vacuumaspirator toremove droplets of ethanol from the walls of thecentrifugebottle. Stand the bottle in an inverted positionuntil no traceof ethanol is visible. At this stage, the pelletshould stillbe damp.

20. Dissolve the damp pellet of nucleicacid in 3 ml of TE (pH8.0).

21. Purify the crude plasmidDNA either by chromatography oncommercial resins (please seePurification of Plasmid DNA by Chromatography)or isopycniccentrifugation in CsCl-ethidium bromide gradients(please seePurification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous GradientsandPurification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous Gradients).

22. Check the structure of the plasmid by restriction enzymedigestion followed by gel electrophoresis.

REFERENCES

1. Godson, G.N. and Vapnek, D. 1973. A simple method for the large-scale purification of X174 RFI supercoiled DNA. Biochim. Biophys. Acta 299: 516–520.

Caution

Chloroform

Caution

Lysozyme

Lysozyme is caustic to mucous membranes. Wear appropriate glovesand safety glasses.

Caution

Phenol:chloroform

Caution

Radioactive substances

Caution

SDS

SDS (Sodium dodecyl sulfate) is toxic, an irritant, and posesa risk of severe damage to the eyes. It may be harmful by inhalation,ingestion, or skin absorption. Wear appropriate gloves and safetygoggles. Do not breathe the dust.

Recipe

EDTA

To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H₂Oto 800 mL of H₂O. Stir vigorously on a magnetic stirrer. Adjustthe pH to 8.0 with NaOH (~20 g of NaOH pellets). Dispense intoaliquots and sterilize by autoclaving. The disodium salt ofEDTA will not go into solution until the pH of the solutionis adjusted to ~8.0 by the addition of NaOH.

Recipe

Lysozyme

Recipe

NaCl

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 mlof H₂O. Adjust the volume to 1 liter with H₂O. Dispense intoaliquots and sterilize by autoclaving. Store the NaCl solutionat room temperature.

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

SDS

Also called sodium dodecyl sulfate or sodium lauryl sulfate.To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-gradeSDS in 900 mL of H₂O. Heat to 68°C and stir with a magneticstirrer to assist dissolution. If necessary, adjust the pH to7.2 by adding a few drops of concentrated HCl. Adjust the volumeto 1 L with H₂O. Store at room temperature. Sterilization isnot necessary. Do not autoclave.

Recipe

STE

10 mM Tris-Cl (pH 8.0)

0.1 M NaCl

1 mM EDTA (pH 8.0)

Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm²)on liquid cycle. Store the sterile solution at 4°C.

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Recipe

Tris-Sucrose

10% (w/v) sucrose

50 mM Tris-Cl (pH 8.0)

Sterilize the solution by passing it through a 0.22-µmfilter, and store it at room temperature. Solutions containingsucrose should not be autoclaved since the sugar tends to carbonizeat high temperatures.

Preparation of Plasmid DNA: Toothpick Minipreparation

2008-04-24T13:13:00.001-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is prepared directly from bacterial colonies pluckedfrom the surface of agar media with toothpicks.

MATERIALS

Antibiotic for plasmid selection

Bromophenol blue solution, 0.4% (w/v)

Cresol red solution (10 mM)

EDTA (0.5 M, pH 8.0)

Ethidiumbromide (10 mg/ml)

KCl(4 M)

NSS solution

Rich broth

Rich broth agar plates

SYBRGold

METHOD

1. Grow bacterial colonies, transformed with recombinant plasmid,on rich agar medium (LB, YT, or SOB) containing the appropriateantibiotic until they are approx. 2-3 mm in diameter (approx.18-24 hours at 37°C for most bacterial strains).

2. Usea sterile toothpick or disposable loop to transfer asmall segmentof a bacterial colony to a streak or patch ona master agarplate containing the appropriate antibiotic. Transferthe remainderof the colony to a numbered microcentrifuge tubecontaining50 µl of sterile 10 mM EDTA (pH 8.0).

3. Repeat Step2 until the desired number of colonies has beenharvested.

4.Incubate the master plate for several hours at 37°C andthen store it at 4°C until the results of the gel electrophoresis(Step 11 of this protocol) are available. Colonies containingplasmids of the desired size can then be recovered from themaster plate.

5. While the master plate is incubating, processthe bacterialsuspensions as follows: To each microcentrifugetube in turn,add 50 µl of a freshly made solution ofNSS. Close thetop of the tubes and then mix their contentsby vortexing for30 seconds.

6. Transfer the tubes to a 70°Cwater bath. Incubate thetubes for 5 minutes and then allowthem to cool to room temperature.

7. To each tube, add 1.5µl of a solution of 4 M KCl.Vortex the tubes for 30 seconds.

8. Incubate the tubes for 5 minutes on ice.

9. Remove bacterialdebris by centrifugation at maximum speedfor 3 minutes at 4°Cin a microcentrifuge.

10. Transfer each of the supernatantsin turn to fresh microcentrifugetubes. Add to each tube 0.5µl of a solution containing0.4% bromophenol blue if thesamples are to be analyzed onlyby agarose gel electrophoresisor 2 µl of 10 mM cresolred if the samples are to be analyzedboth by PCR and by agarosegel electrophoresis. Load 50 µlof the supernatant intoa slot (5 mm in length x 2.5 mm in width)cast in a 0.7% agarosegel (5 mm thick).

11. After the bromophenolblue dye has migrated two-thirds tothree-fourths the lengthof the gel, or the cresol red dye aboutone-half the lengthof the gel, stain the gel by soaking itfor 30-45 minutes ina DNA-staining solution at room temperature.Examine and photographthe gel under UV illumination.

12. If cresol red has beenused at Step 10, analyze the supernatantsby performing PCRas described in The Basic Polymerase Chain Reaction,using theremainder of each sample as a template.

13. Prepare small-scalecultures of the putative recombinantclones by inoculating 2ml of liquid medium (LB, YT, or SOB)containing the appropriateantibiotic with bacteria growingon the master plate.

14.Use the small-scale bacterial cultures to generate minipreparations(please see Preparation of Plasmid DNA by Alkaline Lysis with SDS: MinipreparationorPreparation of Plasmid DNA by Small-scale Boiling Lysis) ofthe putative recombinant plasmids. Analyze the plasmid DNAsby digestion with restriction enzymes and agarose gel electrophoresisto confirm that they have the desired size and structure.

REFERENCES

1. Barnes, W.M. 1977. Plasmid detection and sizing in single colony lysates. Science 195: 393–394.

Caution

Ethidium bromide

Ethidium bromide is a powerful mutagen and is toxic. Consultthe local institutional safety officer for specific handlingand disposal procedures. Avoid breathing the dust. Wear appropriategloves when working with solutions that contain this dye.

Caution

KCl (Potassium chloride)

KCl (Potassium chloride) may be harmful by inhalation, ingestion,or skin absorption. Wear appropriate gloves and safety glasses.

Caution

SYBR Gold

SYBR Gold is supplied by the manufacturer as a 10,000-fold concentratein DMSO which transports chemicals across the skin and othertissues. Wear appropriate gloves and safety glasses and decontaminateaccording to Safety Office guidelines. See DMSO.

Recipe

Cresol red solution

Dissolve 4 mg of the sodium salt of cresol red (Aldrich) in1 ml of sterile H₂O. Store the 10 mM solution at room temperature.

Recipe

EDTA

Recipe

Ethidium bromide

Add 1 g of ethidium bromide to 100 ml of H₂O. Stir on a magneticstirrer for several hours to ensure that the dye has dissolved.Wrap the container in aluminum foil or transfer the 10 mg/mlsolution to a dark bottle and store at room temperature.

Recipe

KCl

For a 1 M solution of KCl, dissolve 74.55 g of KCl in 900 mlof H₂O. Make up the volume to 1L wih H₂O and autoclave for 20minutes on liquid cycle. Store at room temperature.

Ideally, this solution should be divided into small (approx.100 µl) aliquots in sterile tubes and each aliquot thereafterused only once.

Recipe

NSS solution

0.2N NaOH

20% sucrose

0.5%SDS

20% sucrose

This solution should be made fresh for each use. Store the solutionat

room temperature until it is required. Discard any NSS solutionthat

remains unused.

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

SYBR Gold

SYBR Gold (Molecular Probes) is supplied as a stock solutionof unknown concentration in dimethylsulfoxide. Agarose gelsare stained in a working solution of SYBR Gold, which is a 1:10,000dilution of SYBR Gold nucleic acid stain in electrophoresisbuffer. Prepare working stocks of SYBR Gold daily and storein the dark at regulated room temperature.

Preparation of Plasmid DNA: Toothpick Minipreparation

2008-04-24T13:13:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is prepared directly from bacterial colonies pluckedfrom the surface of agar media with toothpicks.

MATERIALS

Antibiotic for plasmid selection

Bromophenol blue solution, 0.4% (w/v)

Cresol red solution (10 mM)

EDTA (0.5 M, pH 8.0)

Ethidiumbromide (10 mg/ml)

KCl(4 M)

NSS solution

Rich broth

Rich broth agar plates

SYBRGold

METHOD

3. Repeat Step2 until the desired number of colonies has beenharvested.

6. Transfer the tubes to a 70°Cwater bath. Incubate thetubes for 5 minutes and then allowthem to cool to room temperature.

7. To each tube, add 1.5µl of a solution of 4 M KCl.Vortex the tubes for 30 seconds.

8. Incubate the tubes for 5 minutes on ice.

9. Remove bacterialdebris by centrifugation at maximum speedfor 3 minutes at 4°Cin a microcentrifuge.

12. If cresol red has beenused at Step 10, analyze the supernatantsby performing PCRas described in The Basic Polymerase Chain Reaction,using theremainder of each sample as a template.

13. Prepare small-scalecultures of the putative recombinantclones by inoculating 2ml of liquid medium (LB, YT, or SOB)containing the appropriateantibiotic with bacteria growingon the master plate.

REFERENCES

1. Barnes, W.M. 1977. Plasmid detection and sizing in single colony lysates. Science 195: 393–394.

Caution

Ethidium bromide

Caution

KCl (Potassium chloride)

KCl (Potassium chloride) may be harmful by inhalation, ingestion,or skin absorption. Wear appropriate gloves and safety glasses.

Caution

SYBR Gold

Recipe

Cresol red solution

Dissolve 4 mg of the sodium salt of cresol red (Aldrich) in1 ml of sterile H₂O. Store the 10 mM solution at room temperature.

Recipe

EDTA

Recipe

Ethidium bromide

Recipe

KCl

For a 1 M solution of KCl, dissolve 74.55 g of KCl in 900 mlof H₂O. Make up the volume to 1L wih H₂O and autoclave for 20minutes on liquid cycle. Store at room temperature.

Ideally, this solution should be divided into small (approx.100 µl) aliquots in sterile tubes and each aliquot thereafterused only once.

Recipe

NSS solution

0.2N NaOH

20% sucrose

0.5%SDS

20% sucrose

This solution should be made fresh for each use. Store the solutionat

room temperature until it is required. Discard any NSS solutionthat

remains unused.

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

SYBR Gold

Preparation of Plasmid DNA by Small-scale Boiling Lysis

2008-04-24T13:11:00.000-07:00

oseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is isolated from small-scale (1-2 ml) bacterialcultures by treatment with Triton X-100 and lysozyme, followedby heating. This method is not recommended for preparing plasmidDNA from strains of E. coli that express endonuclease A (endA⁺strains).

MATERIALS

Antibiotic for plasmid selection

Ethanol

Optional, please see Step 5.

Isopropanol

Lysozyme(10 mg/ml)

Prepare the solution fresh in Tris-Cl (pH 8.0).

Rich medium

STET

Sodiumacetate (3.0 M, pH 5.2)

TE (pH 8.0) containing 20µg/ml RNase A

METHOD

1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth)containing the appropriate antibiotic with a single colony oftransformed bacteria. Incubate the culture overnight at 37°Cwith vigorous shaking.

2. Pour 1.5 ml of the culture intoa microcentrifuge tube. Centrifugethe tube at maximum speedfor 30 seconds at 4°C in a microcentrifuge.Store the unusedportion of the culture at 4°C.

3. Remove the medium bygentle aspiration, leaving the bacterialpellet as dry as possible.

4. Resuspend the bacterial pellet in 350 µl of STET.

5. Add 25 µl of a freshly prepared solution of lysozyme.Close the top of the tube and mix the contents by gently vortexingfor 3 seconds.

6. Place the tube in a boiling water bath forexactly 40 seconds.

7. Centrifuge the bacterial lysate atmaximum speed for 15 minutesat room temperature in a microcentrifuge.Pour the supernatantinto a fresh microcentrifuge tube.

8.Precipitate the nucleic acids from the supernatant by adding40 µl of 2.5 M sodium acetate (pH 5.2) and 420 µlof isopropanol. Mix the solution by vortexing, and then allowthe mixture to stand for 5 minutes at room temperature.

9.Recover the precipitated nucleic acids by centrifugationatmaximum speed for 10 minutes at 4°C in a microcentrifuge.

10. Remove the supernatant by gentle aspiration as describedin Step 3 above. Stand the tube in an inverted position on apaper towel to allow all of the fluid to drain away. Use a Kimwipeor disposable pipette tip to remove any drops of fluid adheringto the walls of the tube.

11. Rinse the pellet of nucleicacid with 1 ml of 70% ethanolat 4°C. Remove all of thesupernatant by gentle aspirationas described in Step 3.

12.Remove any beads of ethanol that form on the sides of thetube.Store the open tube at room temperature until the ethanolhasevaporated and no fluid is visible in the tube (2-5 minutes).

13. Dissolve the nucleic acids in 50 µl of TE (pH 8.0)containing DNase-free RNase A (pancreatic RNase). Vortex thesolution gently for a brief period. Store the DNA at -20°C.

REFERENCES

1. Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.

Caution

Lysozyme

Lysozyme is caustic to mucous membranes. Wear appropriate glovesand safety glasses.

Caution

Sodium acetate (NaOAc)

Sodium acetate (NaOAc), see Acetic acid

Recipe

Lysozyme

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

STET

0.1 M NaCl

5% (v/v) Triton X-100

10 mM Tris-Cl (pH 8.0)

1 mM EDTA (pH 8.0)

Make sure that the pH of STET is 8.0 after all ingredients areadded. There is no need to sterilize STET before use.

Recipe

Sodium acetate

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation

2008-04-24T13:09:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterialcultures by treatment with alkali and SDS.

MATERIALS

Alkaline lysis solution I

For preparations of plasmid DNA that are to be subjected tofurther purification by chromatography (please see Purification of Plasmid DNA by Chromatography),sterile Alkaline lysis solution I may be supplemented just beforeuse with the appropriate volume of 20 mg/ml DNase-free RNaseA (pancreatic RNase) to give a final concentration of 100 µg/ml.

Alkaline lysis solution II

Alkaline lysis solution III

Antibiotic for plasmid selection

Ethanol

Optional, please see Step 5.

Isopropanol

Phenol:chloroform (1:1,v/v)

Rich medium

STE

TE (pH 8.0) containing 20µg/ml RNase A

METHOD

1. Inoculate 10 ml of rich medium (LB, YT, or Terrific Broth)containing the appropriate antibiotic with a single colony oftransformed bacteria. Incubate the culture overnight at 37°Cwith vigorous shaking.

2. Transfer the culture into a 15-mltube and recover the bacteriaby centrifugation at 2000g (4000rpm in a Sorvall SS-34 rotor)for 10 minutes at 4°C.

3.Remove the medium by gentle aspiration, leaving the bacterialpellet as dry as possible.

4. Resuspend the bacterial pelletin 200 µl of ice-coldAlkaline lysis solution I by vigorousvortexing, and transferthe suspension to a microcentrifugetube.

5. Add 400 µl of freshly prepared Alkaline lysissolutionII to each bacterial suspension. Close the tube tightly,andmix the contents by inverting the tube rapidly five times.Donot vortex! Store the tube on ice.

6. Add 300 µlof ice-cold Alkaline lysis solution III.Close the tube anddisperse Alkaline lysis solution III throughthe viscous bacteriallysate by inverting the tube several times.Store the tube onice for 3-5 minutes.

7. Centrifuge the bacterial lysate atmaximum speed for 5 minutesat 4°C in a microcentrifuge.Transfer 600 µl of thesupernatant to a fresh tube.

8.Add an equal volume of phenol:chloroform. Mix the organicandaqueous phases by vortexing and then centrifuge the emulsionat maximum speed for 2 minutes at 4°C in a microcentrifuge.Transfer the aqueous upper layer to a fresh tube.

9. Precipitatenucleic acids from the supernatant by adding600 µl ofisopropanol at room temperature. Mix the solutionby vortexingand then allow the mixture to stand for 2 minutesat room temperature.

10. Collect the precipitated nucleic acids by centrifugationat maximum speed for 5 minutes at room temperature in a microcentrifuge.

11. Remove the supernatant by gentle aspiration as describedin Step 3 above. Stand the tube in an inverted position on apaper towel to allow all of the fluid to drain away. Removeany drops of fluid adhering to the walls of the tube.

12.Add 1 ml of 70% ethanol to the pellet and recover the DNAbycentrifugation at maximum speed for 2 minutes at room temperaturein a microcentrifuge.

13. Remove all of the supernatant bygentle aspiration as describedin Step 3.

14. Remove any beadsof ethanol that form on the sides of thetube. Store the opentube at room temperature until the ethanolhas evaporated andno fluid is visible in the tube (2-5 minutes).

15. Dissolvethe nucleic acids in 100 µl of TE (pH 8.0)containing20 µg/ml DNase-free RNase A (pancreatic RNase).Vortexthe solution gently for a few seconds and store at -20°C.

REFERENCES

1. Birnboim, H.C. and Doly, J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513–1523.[Abstract/Free Full Text]

2. Ish-Horowicz, D. and Burke, J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9: 2989–2998.[Abstract/Free Full Text]

Caution

Phenol:chloroform

Recipe

Alkaline Lysis Solution I

50 mM glucose

25 mM Tris-Cl (pH 8.0)

10 mM EDTA (pH 8.0)

Prepare Solution I from standard stocks in batches of approx.100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm²) onliquid cycle, and store at 4°C.

For plasmid preparation.

Recipe

Alkaline Lysis Solution II

0.2N NaOH (freshly diluted from a 10 N stock)

1%(w/v) SDS

Prepare Solution II fresh and use at room temperature.

For plasmid preparation.

Recipe

Alkaline Lysis Solution III

5 M potassium acetate, 60.0ml

glacial acetic acid, 11.5ml

H₂O, 28.5 ml

The resulting solution is 3 M with respect to potassium and5 M with respect to acetate. Store the solution at 4°C andtransfer it to an ice bucket just before use.

For plasmid preparation.

Recipe

Rich

SOB

For solid medium, pleasesee Media Containing Agar or Agarose.

Recipe

STE

10 mM Tris-Cl (pH 8.0)

0.1 M NaCl

1 mM EDTA (pH 8.0)

Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm²)on liquid cycle. Store the sterile solution at 4°C.

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Phosphorylation of DNA Molecules with Protruding 5'-Hydroxyl Termini

2008-04-24T13:07:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Bacteriophage T4 polynucleotide kinase catalyzes the transferof -³²P of ATP to the terminal 5'-hydroxyl groups of single-or double-stranded nucleic acids. When [-³²P]ATP of high specificactivity (3000-7000 Ci/mmole) is used as a substrate, approximately40-50% of the protruding 5' termini in the reaction become radiolabeled.

MATERIALS

10x Bacteriophage T4 polynucleotidekinase buffer

Ammoniumacetate (10 M)

Optional, please see Step 3.

Bacteriophage T4 polynucleotide kinase

DNA (10-50 pmoles)

The DNA should be dephosphorylated as described in Dephosphorylation of DNA Fragments with Alkaline Phosphataseorsynthesized with a 5'-hydroxyl moiety.

EDTA (0.5 M, pH 8.0)

Ethanol

Optional, please see Step 5.

[-³²P]dATP (10 mCi/ml, sp.act. 3000-7000 Ci/mmole)

METHOD

1. In a microcentrifuge tube, mix the following reagents:

dephosphorylatedDNA		10-50 pmoles
10x bacteriophage T4 polynucleotide kinasebuffer (10-20 units)		5 µl
10 mCi/ml [-³²P]ATP (sp.act. 3000-7000 Ci/mmole)		50 pmoles
bacteriophage T4 polynucleotidekinase		10 units
H₂O		to 50 µl

Incubate the reactionfor 1 hour at 37°C.

Ideally, ATP should be in a fivefoldmolar excess over DNA 5'ends, and the concentration of DNAtermini should be 0.4 µM.The concentration of ATP inthe reaction should therefore be>2 µM, but this israrely achievable in practice. Toincrease the specific activityof the radiolabeled DNA product,increase the amount of [-³²P]ATPused in the phosphorylationreaction. Decrease the volume ofH₂O to maintain a reactionvolume of 50 µl.

2. Terminatethe reaction by adding 2 µl of 0.5 M EDTA(pH 8.0). Measurethe total radioactivity in the reaction mixtureby Cerenkovcounting in a liquid scintillation counter.

3. Separate theradiolabeled probe from unincorporated dNTPsby either:

•	spun-columnchromatography through SephadexG-50
	or
•	conventionalsize-exclusion chromatographythrough 1-ml columns of SepahadexG-50 (equilibrated in TE)
	or
•	tworounds of selectiveprecipitation of the radiolabeled DNA withammonium acetateand ethanol

4. Measure the amount of radioactivity in theprobe preparationby Cerenkov counting. Calculate the efficiencyof transfer ofthe radiolabel to the 5' termini by dividingthe amount of radioactivityin the probe by the total amountin the reaction mixture (Step2).

REFERENCES

1. Wu, G., Jay, E., and Roychoudhury, R. 1976. Nucleotide sequence analysis of DNA. Methods Cancer Res. 12: 87–176.

2. Berkner, K.L. and Folk, W.R. 1977. Polynucleotide kinase exchange reaction: Quantitative assay for restriction endonuclease-generated 5'-phosphoryl termini in DNA. J. Biol. Chem. 252: 3176–3184.[Free Full Text]

Caution

Ammonium acetate

Ammonium acetate H₃CCOONH₄ may be harmful by inhalation, ingestion,or skin absorption. Wear appropriate gloves and safety glasses.Use in a chemical fume hood.

Caution

Radioactive substances

Recipe

Ammonium acetate

To prepare a 10 M solution in 1 liter, dissolve 770 g of ammoniumacetate in 800 ml of H₂O. Adjust volume to 1 liter with H₂O.Sterilize by filtration. Alternatively, to prepare a 100-mlsolution, dissolve 77 g of ammonium acetate in 70 ml of H₂Oat room temperature. Adjust the volume to 100 ml with H₂O. Sterilizethe solution by passing it through a 0.22-µm filter. Storethe solution in tightly sealed bottles at 4°C or at roomtemperature. Ammonium acetate decomposes in hot H₂O and solutionscontaining it should not be autoclaved.

Recipe

Bacteriophage T4 Polynucleotide Kinase Buffer

700 mM Tris-Cl (pH 7.6)

100mM MgCl₂

50mM dithiothreitol

Divide the 10x stock into small aliquots and store frozen at-20°C.

Recipe

EDTA

Dephosphorylation of DNA Fragments with Alkaline Phosphatase

2008-04-24T13:03:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Essentially any protein phosphatase (e.g., bacterial alkalinephosphatase [BAP], calf intestinal phosphatase [CIP], placentalalkaline phosphatase, and shrimp alkaline phosphatase [SAP])will catalyze the removal of 5' phosphates from nucleic acidtemplates. Because CIP and SAP are readily inactivated, theyare the most widely used phosphatases in molecular cloning.Although CIP is cheaper per unit of activity, SAP enzyme hasthe advantage of being readily inactivated in the absence ofchelators.

MATERIALS

Alkaline phosphatase

Chloroform

DNA sample (0.1-10 µg [1-100 pmoles])

Dephosphorylation reactions are usually carried out in a volumeof 25-50 µl containing 1-100 pmoles of 5'-phosphorylatedtermini of DNA.

Dephosphorylation buffer

EDTA (0.5 M, pH 8.0)

EGTA (0.5 M, pH 8.0), ifusing CIP

Ethanol

Optional, please see Step 5.

Phenol:chloroform (1:1,v/v)

ProteinaseK

Restriction enzyme(s)

Please see Step 9.

SDS(10% w/v), if using CIP

Sodiumacetate (3 M, pH 7.0 [if using CIP] and pH 5.2)

TE (pH 7.6)

Tris-Cl (1 M, pH 8.5)

METHOD

1. Use the restriction enzyme of choice to digest to completion1-10 µg (10-100 pmoles) of the DNA to be dephosphorylated.

CIP and SAP will dephosphorylate DNA at a slightly reducedefficiencyin restriction buffers that have been adjusted topH 8.5 with10x CIP or 10x SAP buffer, as is done in the nextstep. If thisis unacceptable, the restricted DNA may be purifiedby extractionwith phenol:chloroform and standard precipitationwith ethanoland then dissolved in a minimal volume of 10 mMTris-Cl (pH8.5).

2. Dephosphorylate the 5' ends of therestricted DNA with eitherCIP or SAP.

To dephosphorylate DNAusing CIP

Add to the DNA:

10xCIP dephosphorylation buffer		5µl
H₂O		to48 µl

Add the appropriate amountof CIP.

1 unit of CIP will dephosphorylateapprox. 1 pmoleof 5'-phosphorylatedtermini (5'-recessed orblunt-ended DNA)or approx. 50 pmolesof 5'-protruding termini.These amountsmay vary slightly fromone manufacturer to thenext.

Incubatethe reaction for 30 minutes at 37°C,add a secondaliquotof CIP, and continue incubation for a further30 minutes.

Toinactivate CIP at the end of the incubationperiod, add SDSand EDTA (pH 8.0) to final concentrations of0.5% and 5 mM,respectively. Mix the reagents well, and addproteinase K toa final concentration of 100 µg/ml. Incubatefor 30 minutesat 56°C.

Alternatively, CIP can be inactivatedby heatingto 65'C for30 minutes (or 75'C for 10 minutes) inthe presenceof 10 mMEGTA (pH 8.0).

IMPORTANT
Use EGTAnot EDTA.

Cool the reaction to room temperature and purifythe DNA byextracting it twice with phenol:chloroform and oncewith chloroformalone.

Proteinase K and SDS used to inactivateand digestCIP mustbe completely removed by extraction withphenol:chloroformpriorto subsequent enzymatic treatments (phosphorylationbypolynucleotidekinase, ligation, etc.).

Glycogen or linearpolyacrylamide can be added as a carrierbefore phenol:chloroformextraction if small amounts of DNA(<100> the reaction. Do not add carrier nucleicacid (tRNA, salmonsperm DNA, etc.), as it will compete withthe dephosphorylatedDNA for the radiolabeled ATP during thekinasing reaction.

To dephosphorylate DNA using SAP

Add to the DNA:

10x SAPdephosphorylation buffer		5 µl
H₂O		to48 µl

Add the appropriate amount of SAP.

1 unit of SAP will dephosphorylateapprox. 1 pmole of 5'-phosphorylatedtermini (3'-recessed or5'- recessed) or approx. 0.2 pmole ofblunt-ended DNA. Theseamounts may vary slightly from one enzymemanufacturer to thenext.

Incubate the reaction for 1 hour at 37°C.

Toinactivate SAP, transfer the reaction to 70°C, incubatefor 20 minutes, and cool to room temperature.

3. Transferthe aqueous phase to a clean microcentrifuge tube,and recoverthe DNA by standard ethanol precipitation in thepresence of0.1 volume of 3 M sodium acetate (pH 5.2) if SAPwas used or0.1 volume of 3 M sodium acetate (pH 7.0) if CIPwas used.

4.Allow the precipitate to dry at room temperature before dissolvingit in TE (pH 7.6) at a DNA concentration of >2 nmoles/ml.

REFERENCES

1. Chaconas, G. and van de Sande, J.H. 1980. 5'-32P labeling of RNA and DNA restriction fragments. Methods Enzymol. 65: 75–85.

Caution

Chloroform

Caution

Phenol:chloroform

Caution

Proteinase K

Proteinase K is an irritant and may be harmful by inhalation,ingestion, or skin absorption. Wear appropriate gloves and safetyglasses.

Caution

SDS

Caution

Sodium acetate (NaOAc)

Sodium acetate (NaOAc), see Acetic acid

Recipe

Alkaline phosphatase

Calf intestinal alkaline phosphatase (CIP)

Shrimp alkaline phosphatase (SAP)

Recipe

Dephosphorylation buffer

CIP dephosphorylation buffer

SAP dephosphorylation buffer

Recipe

EDTA

Recipe

EGTA (ethylene glycol-bis[β-aminoethyl ether]-N,N,N',N'-tetraacetic acid) (1 M)

250 g EGTA (m.w. = 380.4)

53 g NaOH

HCl

Add 250 g of EGTA and 53 g of NaOH to 400 ml H₂O. Adjust thepH to 7.0 with HCl. Adjust total volume to 660 ml with H₂O.Filter, autoclave, and store at 4°C (lasts ~1 mo).

Recipe

Proteinase K

(20 mg/ml) Purchase as a lyophilized powder and dissolve ata concentration of 20 mg/ml in sterile 50 mM Tris (pH 8.0),1.5 mM calcium acetate. Divide the stock solution into smallaliquots and store at -20°C. Each aliquot can be thawedand refrozen several times but should then be discarded. Unlikemuch cruder preparations of protease (e.g., pronase), proteinaseK need not be self-digested before use.

Recipe

SDS

Recipe

Sodium acetate

Recipe

TE buffer, 10X

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)

Sterilize solutions by autoclaving for 20 min at 15 psi (1.05kg/cm²) on liquid cycle. Store the buffer at room temperature.

Recipe

Tris-Cl

Tris base

HCl

To prepare a 1 M solution, dissolve 121.1 g of Tris base in800 mL of H₂O. Adjust the pH to the desired value by addingconcentrated HCl.

pH	HCl

7.4	70 mL
7.6	60 mL
8.0	42mL

Allow the solution to cool to room temperature before makingfinal adjustments to the pH. Adjust the volume of the solutionto 1 L with H₂O. Dispense into aliquots and sterilize by autoclaving.

If the 1 M solution has a yellow color, discard it and obtainTris of better quality. The pH of Tris solutions is temperature-dependentand decreases ~ 0.03 pH units for each 1°C increase in temperature.For example, a 0.05 M solution has pH values of 9.5, 8.9, and8.6 at 5°C, 25°C, and 37°C, respectively.

Drosophila Cell Culture and Transformation

2008-04-24T12:59:00.000-07:00

Lucy Cherbas and Peter Cherbas

Adapted from "Drosophila Cell Culture and Transformation," Chapter 20, in Drosophila Protocols (eds. Sullivan et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.

INTRODUCTION

Permanent Drosophila cell lines derived from mixed embryonictissues including the most commonly used lines, S2 and Kc, havebeen available for ~30 yr. More recently, lines derived fromspecific tissues, imaginal discs, and the larval central nervoussystem have come into use. Although cultured cells were originallyused by Drosophilists mainly as convenient sources of DNA orcarrier RNA, that situation has changed, as an armamentariumof techniques for using the cells has slowly but steadily evolved.Most investigators use Drosophila cell lines as hosts for transformationexperiments. The goal may be to characterize a promoter, toinvestigate the role of a transcription factor, to overexpressa polypeptide, or to do something more novel. This article providesan organized collection of pointers to published protocols.

BACKGROUND

Cherbas and Cherbas (1998) provides a brief account of the historiesof Drosophila cell lines with speculations on the histologicalorigin(s) of the embryonic lines and a tabular account of geneexpression in various lines. Broader biological reviews of thelines are also available (Schneider and Blumenthal 1978; Cherbas and Cherbas 1981;Sang 1981; Echalier 1997). For cell maintenance and cloningprotocols, see Cherbas and Cherbas (1998), and for transformationprotocols, see Cherbas et al. (1994), supplemented, for theS2 expression system, by Kirkpatrick and Shatzman (1999).

It is important to appreciate that although the many embryoniccell lines are similar in properties, they are not identical.For example, S2 cells respond very poorly to the hormone ecdysone,but Kc cells exhibit a vigorous response. The first step isto choose a cell line that meets one’s requirements. Whilethere is as yet no central repository for Drosophila cell lines,S2 and Kc cells are widely used. The newer lines can generallybe obtained from their originators. Specific lines of interestcan be identified with an inquiry to http://www.bio.net:80/hypermail/DROS/.

A second step is to understand the virtues and limitations ofthe various transformation procedures available. Transient expressionprovides a quantitative assessment of reporter gene expression.Cotransformation with expression vectors for other genes allowsthe assessment of their effects on the reporter gene’sexpression. Transient expression is fast and statistically meaningful,as one is observing average behavior for a population of cells.However, only a minority of cells express; hence, one cannotlook for losses of function or determine much about titers (e.g.,of a coexpressed transcription factor) in the relevant population.

In contrast, stable transformation generates cells containinghigh- (>1000) or low-copy numbers of the vector in question.Either can be accomplished using either illegitimate recombinationor transposition. High-copy numbers are of interest principallyas a way to overexpress proteins. Usually one uses S2 cells,in which illegitimate recombination leads directly to long chromosomaldirect repeats (arrays) containing the gene in question. Whenthe gene is not toxic, this expression system is simple andrelatively fast. For a detailed account of applications, seeKirkpatrick and Shatzman (1999). Because of interactions withinthe array, these are not useful for gene-regulation studies.Transposition can also be used to generate high-copy numbers,but there is no published background.

Low-copy-number transformants can also be generated by illegitimaterecombination, particularly in Kc cells. A better procedureis P-element-mediated transposition, which can readily yieldsingle-copy transformants. In either case, one selects transformedcells using a selectable marker and works either with the populationor with specific clones. The advantages of this procedure arestable expression in the entire population and the ability toquantify expression levels when working with clones. The disadvantagesinclude the time involved; cloning requires 3-6 wk, dependingon the health of the transformed cells. In addition, individualclones can vary in ways that are unrelated to the transformation,and thus a collection of clones must be studied.

Parahomologous targeting occurs at high frequency when Drosophilacells are transformed with a plasmid containing 5-10 kb of chromosomalhomology. It reveals itself as illegitimate insertions (containingvector sequences) within a few kilobases of the target site.This can be useful as a way to knock out genes.

REQUIREMENTS FOR CELL CULTURE

Culture Media and Washing Solutions

For a detailed discussion of culture media, see Cherbas and Cherbas (1998).Most common media for Drosophila cell culture are now commerciallyavailable (e.g., M3 medium supplemented with 10% fetal calfserum). Antibiotics are not in general necessary. Also, someof the commercial media designed for lepidopteran cells worksatisfactorily for Drosophila cells. For example, the HyCloneproduct CCM-3 is a convenient medium for Kc cells. It is particularlyattractive in that its (proprietary) formulation obviates theneed for serum. However, ecdysone-induced gene expression isunreliable in CCM-3. No one medium is universally best, andit is important to test each medium for precisely those experimentsit is intended to serve. Media must be prepared using the highestpurity water, e.g., Milli-Q purified or double-distilled water.Avoid building distilled water that has been transported throughmetal pipes, at least for long-term work. Low-grade medium toxicity,whatever its cause, can take days or weeks to develop and evenlonger to diagnose; it is better to simply avoid foreseeableproblems. To wash cells, we recommend the use of

Robb’s minimal saline(Robb 1969).

General Laboratory Practices

Although culture methods for Drosophila cells are quite forgiving,successful work requires disciplined laboratory hygiene, routinemaintenance techniques to select for desirable or undesirableproperties, and sterile conditions. Maintaining sterility ismore challenging for cell culture than for bacteriology. Themedium is very rich and the cells of interest grow much moreslowly than most likely contaminants. Although some investigatorsmanage to maintain cells for short periods at the laboratorybench, the best solution is to use a laminar flow hood and adopthigh standards of sterile technique. Because Drosophila cellsdo not ordinarily harbor human pathogens, it is acceptable towork in a pharmacological laminar flow hood rather than in themore awkward (and more expensive) biosafety cabinet.

Equipment

Drosophila cells grow in an air atmosphere; a CO₂ incubatoris not required. Cultures can be grown in Petri dishes (tissue-culturegrade) stored in air-tight, plastic food-storage containersto maintain high humidity. Because they grow optimally at 25°C,it is possible to maintain Drosophila cells on the bench top.Be aware that some building temperature-control systems areerratic enough to induce heat shock.

Spinner flasks are generally used for large-volume cultures.Since even a single bacterial or fungal spore leads to substantialwaste of both medium and time, special precautions should betaken to sterilize these flasks. Fill the spinner with cleanwater. Autoclave, and leave at room temperature for 24 h. Autoclaveagain. Pour off the water before use. Double autoclaving issufficient to eliminate rare spores whose germination is inducedby heat. The water leaches out chemical contaminants (e.g.,soap residue) and detectably improves the health of the cultures.Cells grow somewhat more slowly in spinner flasks and appearmore battered than in stationary culture. It is important notto overfill spinner flasks; use no more than half the nominalcapacity. The stirring bar should turn just quickly enough tomaintain the cells in suspension, usually ~60 rpm. We maintaincell lines in Petri dishes and use spinners only to expand populations.

Kc and S2 cells grow exponentially between ~5 x 10⁵ and 10⁷cells/mL and are healthiest if kept within this range. The cellsdouble approximately every 24 hs, which generally means dilutingthem 10-fold every 3-4 d. Cell density is easily determinedusing a standard hemocytometer. Most Drosophila lines are eithernonadherent or adhere to the substrate loosely enough so thatthey can be dislodged by blowing medium at the surface witha Pasteur pipette. Trypsinization is usually not required. Formost purposes, it is adequate to simply dilute an aliquot ofa dense culture in fresh medium. If desired, cells can be pelletedby gentle centrifugation (~700g for 2 min), and then resuspendedby pipetting in fresh medium.

Drosophila cells appear to be much less subject to gross changesduring culture than are mammalian cells; the karyotype is generallystable, and changes in cell properties with time in cultureare usually subtle. Nevertheless, changes do occur. To minimizeproblems, ampoules of frozen cells can be maintained from whichto restart incubator cultures approximately every 6 mo (Cherbas et al. 1994;Cherbas and Cherbas 1998).

PREPARATION OF DNA AND RNA

Drosophila tissue culture cells present no special problemsfor the isolation of DNA or RNA. RNA preparations (Chomczynski and Sacchi 1987)generally yield about 200 µg of total RNA/10⁷ Kc cells.DNA preparations by the method of Maniatis et al. (1982) typicallyyield ~5 µg/10⁷ Kc cells.

CLONING CELLS

Any Drosophila cell line can be cloned by limiting dilution(in 96-well plates) provided the growth of isolated cells issupported by the presence of a suspension of X-rayed feedercells. Some lines (notably S2) can be cloned in soft agar, ata considerable saving of labor and money (Cherbas et al. 1994;Cherbas and Cherbas 1998).

TRANSFECTION METHODS

A number of techniques are available for the introduction ofexogenous DNA into cultured Drosophila cells (Cherbas et al. 1994;Cherbas and Cherbas 1998). Each method causes at least somedisruption to the normal physiology of the cells. In our experience,this is more of a problem with calcium phosphate than with electroporation.It is therefore a good idea to check the effect of transfectionitself on the physiological process being studied.

Calcium Phosphate-DNA Coprecipitation

This technique is the oldest and probably the most commonlyused. It requires no special equipment and is very inexpensive.However, it works for only some cell lines and it requires afixed amount of DNA (20 µg/mL precipitate), thereby placinglimits on experimental design.

Electroporation

This technique is much less labor-intensive than calcium phosphate-DNAcoprecipitation. It can be used to introduce a variety of moleculesinto cells, including double-stranded RNA and proteins. Thetechnique confers no apparent restrictions on the amount ofDNA being transfected and appears to work on all cell lines.It does, however, require relatively expensive equipment. Also,the parameters for shocking the cells must be optimized foreach cell line.

The electroporation protocol for Kc cells (Cherbas et al. 1994)can be modified for S2 cells by increasing the voltage from440 V/cm (Kc) to 715 V/cm (S2) (Cherbas and Cherbas 1998). Thehigher voltage also works well for S3 cells, for the shibireline EH34A3, and for the haploid line D (K. Klueg and M.A.T.Muskavitch, pers. comm.).

Lipofection

Like electroporation, this technique apparently works for allDrosophila cell lines and permits the introduction of a widevariety of types and concentrations of molecules. It requiresno special equipment, but the reagents are relatively expensive.Commercial lipofection systems work well with Drosophila lines(e.g., Søndergaard 1996).

TRANSIENT EXPRESSION

Transient expression is a simple, rapid procedure for examiningthe expression of exogenous DNA in cultured cells. DNA is introduced(by any of the transfection procedures described above), andits expression is monitored soon thereafter in the heterogeneouspopulation of transfected cells. Typically, levels of expressionin individual cells vary over a wide range, with a large fractionof the expression due to a small fraction of the cells. Thefollowing issues should be considered in designing a transientexpression experiment:

Reporters

The reporters used most commonly for mammalian cells, Escherichiacoli ß-galactosidase (lacZ), chloramphenicol acetyltransferase(CAT), and firefly luciferase, also work for Drosophila cells.ß-galactosidase is easily assayed without specialequipment, but background Drosophila ß-galactosidaseactivity limits the sensitivity of the assay. The endogenousenzyme is particularly a problem in studies of ecdysone responses,because the Drosophila gene is induced by the hormone. CAT isa useful reporter because there is no background activity and,like lacZ, it can be assayed without special equipment. However,the reagents for CAT assays are expensive and the assay is labor-intensive.Luciferase is a very sensitive assay with no background enzymaticactivity and is very easily assayed. It does, however, requireaccess to a luminometer. Green fluorescent protein (GFP) isuseful for some purposes. It is readily detected in transfectedcells, but its expression is less readily quantifiable.

Experimental Design

In designing transient expression experiments, it is importantto bear in mind that most cells will express the exogenous geneproduct at low levels or not at all. However, because cotransfectionfrequencies are very high, there are many successful strategiesfor using transients; these depend on ways of observing onlythe transfected cells. Although techniques exist for isolatingtransfected cells (e.g., cell sorting based on a cotransfectedmarker), the more common strategy is to use an identifiablereporter that can be detected by immunostaining, by its intrinsicfluorescence (e.g., GFP), or by enzymatic assay. Because ofthe high efficiency of cotransfection, one can readily examinethe effects of the product of one plasmid on the expressionfrom a second plasmid.

Promoters

To achieve a high level of expression of the protein of interest,investigators express the gene from a strong constitutive Drosophilapromoter such as actin5C or ubiquitin (the constitutive copiapromoter is also useful, but is much weaker than act5C or ubiquitin).A number of vectors all make use of an act5C promoter and havebeen used extensively for expression in Kc or S2 cells. pPac(Krasnow et al. 1989), for example, permits cloning of an openreading frame between an act5C promoter and an act5C polyadenylationregion. Likewise, Ract-Hadh (Swevers et al. 1996) was made bysubstituting an actin5C promoter fragment for the metallothioneinpromoter of pRmHa-1; pRmHa-1 contains a metallothionein promoter,followed by a polylinker and a polyadenylation region from Adh(Bunch et al. 1988). pCMA (Hu 1998) was made by adding an act5Cpromoter fragment to the expression vector pCMX. The resultingplasmid contains (in order) an actin5C promoter, a T7 promoter,a cytomegalovirus (CMV) promoter, a polylinker, and a polyadenylationregion from SV40, and can be used for expression in Drosophilacells, in mammalian cells, and in vitro.

Quantification

Quantification is made easier if an internal control is usedfor transfection efficiency. The internal control is usuallya second plasmid carrying a second reporter expressed from aconstitutive promoter, which is included at a constant concentrationin each transfection. The same issues apply as for choosingthe primary reporter. A very useful combination is firefly luciferaseas the primary reporter and luciferase from the coelenterateRenilla as the secondary reporter. Reagents for assaying thetwo luciferases in succession from a single extract are commerciallyavailable.

Effect of Culturing Conditions on Levels of Expression

Treatment of the cells during the experiment can affect thelevel of a reporter in unexpected ways. For example, CAT activity,expressed from a constitutive promoter, is reduced ~30% by ecdysonetreatment. Firefly luciferase activity is elevated about threefoldby ecdysone, and Renilla luciferase is elevated about fivefold.These may be indirect effects of the hormone on stability ofproteins or RNAs, or possibly on translational efficiency. Itis advisable to check the properties of the reporter expressedfrom a constitutive promoter under the conditions of the experiment,and to correct the experimental results accordingly.

STABLE TRANSFORMATION

Strategies

General Considerations
Drosophila cell lines may be stably transformed in several ways;the technique used should be chosen to fit the investigator’spurposes. A traditional procedure, simple transfection witha supercoiled plasmid or combination of plasmids, leads to thegeneration of long head-to-tail arrays of the plasmid(s) insertedinto the chromosome. In Kc cells, these arrays are fairly small(typically, 1-10 copies). In S2 cells, they are immense (>>1000copies). This procedure is simple and can work well for makingcell lines that produce large quantities of an exogenous protein.It is clear that reporter expression is not linear with copynumber at higher levels. It is possible that silencing and/orheterochromatization occurs. Two recent alternative procedureslead to the insertion of small numbers of copies of the plasmidsequence, either in random positions (by transposition) or ina targeted region (by parahomologous targeting).

Stably transformed populations must be selected following transfection;the procedure is relatively simple and rapid. However, it isoften better to clone the transfected cells in the presenceof the selective agent, rather than merely selecting a resistantpopulation. There is wide variation in the properties of transformedcells resulting from a single transfection, including numberof plasmid copies incorporated, expression per plasmid copy,and extent of both basal expression and inducibility of an induciblepromoter. Bulk selection leads to a population consisting ofthose transformants that grow most rapidly, whereas those transformantswhose properties are most desirable for the experiment may belost. The ease of finding transformed cells with suitable propertiesvaries greatly with the protein being expressed. In some cases(e.g., CNN4, Fig. 1B-E), a bulk-selected population is quitesatisfactory, whereas in others (e.g., N, Fig. 1A) cloning isessential.

Figure 1. Examples of the uses of stably transformed S2 cell lines. (A) Mixed aggregate of cells from two stably transformed clonal lines, one transformed with a plasmid expressing Delta (Dl) from a metallothionein (Mt) promoter and the other transformed with a plasmid expressing Notch (N) from a Mt promoter. Cells from the two lines were mixed, treated with Cu⁺⁺, and immunostained for Dl (green) and N (red). Untransformed cells express no detectable N or Dl. The arrow indicates a vesicle within the N-expressing cell, which has taken up Dl protein. (B-E) Effect of high-level expression in mitotic S2 cells of the testis-specific centrosomin product CNN4 (Li et al. 1998). Stably transformed populations of S2 cells were selected following transfection with either the methotrexate resistance plasmid p8HCO alone (B,C) or p8HCO together with a plasmid in which CNN4 is expressed from a Mt promoter (D,E). (C,E) Cells were treated with Cu⁺⁺ to induce the Mt promoter. Cells were stained for centrosomes (red), tubulin (green), and DNA (blue). Untransformed cells, without or with Cu⁺⁺ treatment, are indistinguishable from the pattern seen in B, C, and D. Induction of the CNN4 protein leads to a proliferation of centrosomes and disorder of the tubulin array. (A is courtesy of K. Klueg and M. Muskavitch and reproduced from Klueg et al. 1998, with permission by the American Society for Cell Biology © 1998. B-E were kindly provided by T. Megraw.)

Promoters
The selectable marker must be expressed from a promoter whosestrength is adequate to render the cells resistant to the selectiveagent. When transformation is expected to lead to the generationof long arrays, a relatively weak promoter may be adequate.Thus, for example, a methotrexate-resistant dihydrofolate reductase(DHFR) expressed from a copia promoter confers complete methotrexateresistance to S2 cells when present in at least 10 copies/haploidgenome (Moss 1985; Cherbas et al. 1994). S2 cells transformedby array formation typically contain ~1000 plasmid copies perhaploid genome. On the other hand, transposition and parahomologoustargeting typically result in a small number of copies (1-5)of the plasmid sequence per cell. These procedures require thatthe methotrexate resistance marker be expressed from a strongerpromoter, such as the actin5C promoter.

When a plasmid that results in the expression of an exogenousprotein is stably introduced, it is often necessary to regulatethe expression of that protein with an inducible promoter tominimize its toxic effects while the line is maintained. Thisis usually done with a copper-inducible metallothionein (Mt)promoter, for which a number of vectors are available (Bunch et al. 1988).Figure 1 shows some examples of Cu⁺⁺-induced expression of exogenousproteins from the Mt promoter. It is important to realize thatthere is a low level of basal expression from the Mt promoterin cells that have not been treated with Cu⁺⁺. Typically, theinduction by Cu⁺⁺ is ~30-fold (Bunch et al. 1988). Both thebasal level of expression and the extent of the induction mayvary greatly from clone to clone for a given transfection.

When stably transformed cells are to be used for large-scaleproduction of an exogenous protein, it is often helpful if theprotein is secreted into the cell membrane or into the medium(see, e.g., Johanson et al. 1995). If the protein in questionis not normally a secreted protein, its coding sequence canbe cloned into one of a number of commercially available vectorsthat add a signal peptide to the expressed protein (Kirkpatrick and Shatzman 1999).

Selectable Markers

The most commonly used selective systems for stable transformationof Drosophila cells are methotrexate, hygromycin B, and -amanitin.Methotrexate resistance can be conferred by multiple copiesof the DHFR resistance gene expressed from a copia promoter,or a single copy expressed from an actin promoter. Resistanceto -amanitin can be conferred by a single copy of a genomicfragment containing a resistant form of the RNA polymerase subunitRPII215 (Jokerst et al. 1989; Segal et al. 1996); multiple copiescause no apparent toxicity (Thomas and Elgin 1988).

When cells are transformed by array formation, it is not necessaryto incorporate the selectable marker into the plasmid of interest;cotransfection of two plasmids always leads to incorporationof both plasmids into a single array (see Cherbas et al. 1994).Indeed, it is preferable to use cotransfection. Not only doesthis approach minimize the labor in plasmid construction, italso permits more control over the number of plasmid copiesincorporated in the transformed cells, since the arrays generallycontain the cotransfected plasmids in the same proportions thatwere present in the mixture used for transfection.

Selection Procedures

Following transfection, allow 2 d for the cells to recover fromthe trauma of transfection and to express the selectable markerbefore adding the selective agent. Killing by the selectiveagent is always slower than it would be in an untransfectedpopulation, because a large number of cells that are not stablytransformed nevertheless express the selective marker transiently.Procedures for methotrexate and amanitin selection, the twosystems with which we are experienced, are given below. Fora detailed protocol for hygromycin selection of S2 cells, seeKirkpatrick and Shatzman (1999).

Methotrexate Selection
Two days after transfection, methotrexate is added to a finalconcentration of 2 x 10^-7 M. If the cells are to be selectedas a population, simply pellet the cells by centrifugation every4 d and resuspend in fresh medium containing methotrexate. Aftera few days, cell proliferation should be noticeably slowed.After ~1 wk, untransformed cells should begin to lyse. If thecells are to be cloned, allow 4 d of selection before cloning,during which growth of methotrexate-sensitive cells largelyceases. Clone the cells in the presence of methotrexate. S2cells can be cloned in soft agar, and Kc cells cloned by dilutionin 96-well plates. For cloning in 96-well plates, it is necessaryto dilute the cells so that there will be approximately oneviable cell per 10 wells. This is obviously a problem when thereis no estimate of the frequency of transformants in the partiallyselected population. Therefore, it is advisable to clone a seriesof 10-fold dilutions of the transfected cells (10² to 10⁵ intactcells/mL) and simply choose plates with an appropriate densityof clones from which to pick transformants for further growth.Methotrexate-resistant lines that contain plasmid arrays shouldbe maintained in the continuous presence of methotrexate asa precaution against the (surprisingly rare) event in whichthe array is lost by homologous recombination.

-Amanitin Selection
-Amanitin kills sensitive cells much more rapidly than doesmethotrexate. As this reagent is both expensive and toxic, continuousselection is usually not an option. Instead, -amanitin is added2 d after transfection (final concentration 5 µg/mL forS2 cells and 10 µg/mL for Kc cells), and the cells areleft undisturbed for 1 wk. By this time, all of the sensitivecells have lysed. It may therefore be difficult to see the resistantcells in the debris of lysed cells. The cells are then clonedor grown up as a population (exactly as for methotrexate selection),except that the selective agent is not added to the medium usedafter the initial 1 wk of selection. It is a good idea to addamanitin to the medium occasionally when maintaining cells containingarrays of plasmid, but it is not necessary (and for most labs,prohibitively expensive) to use continuous amanitin selection.

Arrays

To generate arrays of plasmid, transfect cells by calcium phosphate-DNAcoprecipitation using 1 mL of precipitate for 3 mL of cells.The precipitate contains 20 µg of supercoiled plasmidDNA/mL, a mixture of the plasmid of interest, and a plasmidcarrying a selectable marker. For S2 cells, if the compositionof the plasmids does not lead to selection against their presence,incorporation of ~1000 plasmid copies per haploid genome willresult, in the form of arrays containing the same ratio of plasmidsthat was present in the input mix. The products of this transformationprocedure in other cell lines are less completely characterized.

Transposition

P-element transposition in Kc cells has been described (Segal et al. 1996).For this purpose, both the selectable marker and the fragmentof interest must be included in a single transposon, becausemost cells will incorporate a single transposon (or at most,two to five transposons). Segal et al. (1996) used the plasmidpUChS2-3 as a source of transposase, in which a partially splicedtransposase transcript is expressed from a hsp70 promoter. Usingthe basal expression of the heat-shock promoter (i.e., no heatshock), electroporation of Kc cells with a mixture of 1-2 µgof the transposase plasmid and 11 µg of a 4.2-kb plasmidcontaining the transposon led to incorporation of an averageof two to three copies of the transposon per cell. Virtuallyall of the transformation occurred by P-element transposition,as shown by the absence of flanking vector sequences from thetransformed cells.

Targeting by Parahomologous Recombination

Parahomologous targeting refers to clustered illegitimate recombinationevents that occur at high frequencies in Drosophila cells inthe vicinity of a target sequence. This may simply reflect highlocal concentrations of exogenous DNAs caused by the efficientpairing of plasmid DNAs with their chromosomal homologs. Itis important to point out that such parahomologous events appearto occur during all Drosophila cell transformations. Where theobject is to obtain low-copy-number transpositions, these eventscan represent a serious problem. Still, parahomologous eventsare valuable for other purposes. For example, they can be usedto inactivate a target locus that is functionally haploid inthe targeted line; the ploidy of a gene of interest is not alwaysobvious from either karyotype or Southern analysis. Parahomologoustargeting has been used successfully to generate a Kc line thatis deficient in EcR function; Kc cells are diploid for chromosomeIII (on which EcR is located), but they contain four copiesof EcR, of which only one is functional (Cherbas and Cherbas 1997).High frequencies of rearrangements in the targeted region (onthe order of 50% of transformed clones) can be achieved. A linearfragment is prepared that contains both a selectable marker(methotrexate-resistant DHFR expressed from an actin5C promoter)and at least 4 kb of homology with the targeted chromosomalregion. The DNA is introduced into Kc cells by electroporation;similar results were obtained using 2-60 mg of DNA per electroporationof fragments 8-13 kb in length. The resulting transformantsare cloned in the presence of methotrexate, and the status ofthe targeted region is assessed by Southern analysis and/orphenotypic analysis.

RNA INTERFERENCE

RNA interference has been shown to effectively abolish expressionof genes in several Drosophila cell lines. Worby et al. (2001)have developed a simple protocol for its use and reported successin blocking expression of each (of 10) signal transduction pathwaygenes tested.

REFERENCES

Bunch, T.A., Grinblat, Y., and Goldstein, L.S.B. 1988. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16: 1043–1061.

Cherbas, L. and Cherbas, P. 1981. The effects of ecdysteroid hormones on Drosophila melanogaster cell lines. Adv. Cell Culture 1: 91–124.

Cherbas, L. and Cherbas, P. 1997. "Parahomologous" gene targeting in Drosophila cells: An efficient, homology-dependent pathway of illegitimate recombination near a target site. Genetics 145: 349–358.

Cherbas, L. and Cherbas, P. 1998. Cell culture. In Drosophila: A practical approach (ed. D.B. Roberts), 2nd ed. IRL Press, Oxford, UK.

Cherbas, L., Moss, R., and Cherbas, P. 1994. Transformation techniques for Drosophila cell lines. Methods Cell Biol. 44: 161–179.

Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.

Echalier, G. 1997. Drosophila Cells in Culture. Academic Press, San Diego, CA.

Hu, X. 1998. "The mechanisms of activating the functional ecdysone receptor complex." Ph.D. thesis, Indiana University, Bloomington, IN.

Johanson, K., Appelbaum, E., Doyle, M., Hensley, P., Zhao, B., Abdel-Mequid, S.S., Young, P., Cook, R., Carr, S., Matico, R., et al. 1995. Binding interactions of human interleukin 5 with its receptor subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins. J. Biol. Chem. 270: 9459–9471.

Jokerst, R.S., Weeks, J.R., Zehring, W.A., and Greenleaf, A.L. 1989. Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila. Mol. Gen. Genet. 215: 266–275.

Kirkpatrick, R.B. and Shatzman, A. 1999. Drosophila S2 system for heterologous gene expression. In Gene expression systems (eds. J.M. Fernandez and J.P. Hoeffler), pp. 289–330. Academic Press, San Diego, CA.

Klueg, K., Parody, T., and Muskavitch, M.A.T. 1998. Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol. Biol. Cell. 9: 1709–1723.

Krasnow, M.A., Saffman, E.E., Kornfeld, K., and Hogness, D.S. 1989. Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells. Cell 57: 1031–1043.

Li, K., Xu, E.Y., Cecil, J.K., Turner, F.R., Megraw, T.L., and Kaufman, T.C. 1998. Drosophila centrosomin protein is required for male meiosis and assembly of the flagellar axoneme. J. Cell Biol. 141: 455–467.

Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Moss, R.E. 1985. "Analysis of a transformation system for Drosophila tissue culture cells." Ph.D. thesis, Harvard University, Cambridge, MA.

Robb, J.A. 1969. Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media. J. Cell Biol. 41: 876–885.

Sang, J.H. 1981. Drosophila cells and cell lines. Adv. Cell Culture 1: 125–182.

Schneider, I. and Blumenthal, A.B. 1978. Drosophila cell and tissue culture. In The genetics and biology of Drosophila (eds. M. Ashburner and T.R.F. Wright), Vol. 2a, pp. 265–316. Academic Press, London.

Segal, D., Cherbas, L., and Cherbas, P. 1996. Genetic transformation of Drosophila cells in culture by P element-mediated transposition. Somat. Cell Mol. Genet. 22: 159–165.

Søndergaard, L. 1996. Efficiency of different lipofection agents in Drosophila S-2 cells. In Vitro Cell. Dev. Biol. Anim. 32: 386.

Swevers, L., Cherbas, L., Cherbas, P., and Iatrou, K. 1996. Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor. Insect Biochem. Mol. Biol. 26: 217–221.

Thomas, G.H. and Elgin, S.C.R. 1988. The use of the gene encoding -amanitin-resistant subunit of RNA polymerase II as a selectable marker in cell transformation. Drosophila Inf. Serv. 67: 85.

Worby, C.A., Simonson-Leff, N., and Dixon, J.E. RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci. STKE 2001: pl1. doi: 10.1126/stke.2001.95.pl1.

Recipe

Robb’s minimal saline

Solution A
Reagent	Amount to add to make 1 L
NaCl	3.04g
KCl	2.98 g
Glucose	1.80g
Sucrose	34.23 g
MgSO₄•7H₂O	0.28g
MgCl₂•6H₂O	0.25g
CaCl₂•2H₂O	0.15g
H₂O	to 1 L

Solution B
Reagent	Amount to add to make1 L
Na₂HPO₄•2H₂O	0.356g
KH₂PO₄	0.050 g
HCl(1 N)	adjust to pH 6.75
H₂O	to 1 L

Autoclave solutionsA and B separately; when cool, mix in a 1:1 ratio.

Vectors and Agrobacterium Hosts for Arabidopsis Transformation

2008-04-24T12:56:00.000-07:00

Detlef Weigel and Jane Glazebrook

Adapted from "How to Transform Arabidopsis," Chapter 5, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediatedtransfer of T-DNA. A. tumefaciens is a soil-dwelling bacteriumthat transforms normal plant cells into tumor-forming cellsby inserting a piece of bacterial DNA (the transfer, or "T,"DNA) into the plant cell genome. The T-DNA, which is flankedby left- and right-border (LB and RB) sequences, resides ona tumor-inducing (Ti) plasmid. The Ti plasmid also carries manyof the transfer functions for mobilizing the T-DNA. This articleprovides a brief discussion of the principles of T-DNA transformation,including consideration of T-DNA vectors and their hosts.

RELATED INFORMATION

For protocols describing the transformation of Arabidopsis withAgrobacterium, see In Planta Transformation of Arabidopsis andRoot Transformation of Arabidopsis.

T-DNA Vectors

Numerous T-DNA vectors (see Table 1) and bacterial hosts arenow available and the choice among them will depend on the application.For an excellent discussion of the history of T-DNA vectorsand comparison of many different systems, see the review byHellens et al. (2000b).

The original T-DNA vectors were clumsy, requiring recombinationof the foreign DNA with the resident Ti plasmid in Agrobacterium.It was later discovered that, with the exception of the T-DNAborders, the transfer functions of the Ti plasmid did not needto be present in cis. This discovery led to the developmentof the so-called binary vector systems, in which the Agrobacteriumhost contains a disarmed Ti plasmid. The disarmed plasmid encodesthe transfer functions, but it does not harbor the T-DNA segmentthat will be transferred to the plant cell. Instead, the T-DNAresides on a separate plasmid, which is typically manipulatedin Escherichia coli and then transferred to the Agrobacteriumhost by electroporation (see Transformation of Agrobacterium Using Electroporation)or by direct transformation (see Transformation of Agrobacterium Using the Freeze-Thaw Method).Previously, plasmids were mobilized by triparental mating, butthis method is no longer common.

Older T-DNA vectors, such as pBIN19 (Bevan 1984), have largelyfallen out of favor because of their low copy number in E. coli,which makes it difficult to obtain large amounts of DNA duringvarious cloning steps. More recently developed vectors typicallycontain a high-copy-number origin of replication for E. coli.Another disadvantage of earlier vectors, such as pBIN19, isthat the plant resistance marker is next to the right border.Because T-DNA transfer is directional, with the right borderbeing transferred first, it is better to have the resistancemarker next to the left border to ensure that resistant plantshave received a complete (or nearly complete) copy of the T-DNA.

Additional considerations when choosing a vector include theresistance marker in bacteria, resistance marker in plants,the size of the vector, the presence of a lacZ -peptide-codingsequence surrounding the cloning site (for blue-white selectionin E. coli), and finally the configuration of unique restrictionsites available for cloning. The most common bacterial resistancemarkers are kanamycin, streptomycin or spectinomycin, gentamycin,and tetracycline. Because Agrobacterium strains typically containresistance markers on the chromosome and/or the Ti plasmid (toselect against other bacteria and for the Ti plasmid, respectively),care must be taken to use compatible vector/Agrobacterium combinations.Most Agrobacterium strains (C58C1 and GV3100 are exceptions)carry rifampicin resistance on the chromosome. GV3101 (pMP90)has gentamycin resistance on the Ti plasmid; GV3101 (pMP90RK)carries gentamycin and kanamycin resistance. Vectors that requireselection for tetracycline resistance should not be used withGV3101, because mutants with resistance to 5 µg/ml tetracyclinearise at very high frequency. Note that some T-DNA vectors containonly the replication origin (oriV) for Agrobacterium and thatthe replication functions must be provided in trans by the appropriateTi helper plasmid. These vectors need a helper such as pMP90RK(not to be confused with pMP90).

With respect to plant resistance markers, several families ofT-DNA vectors, such as the pSLJ (Jones et al. 1992), pPZP (Hajdukiewicz et al. 1994),pCAMBIA (http://www.cambia.org.au), and pGreen (Hellens et al. 2000a)series, include members conferring different resistances. Thiscan often be convenient, because it allows retransformationof a plant that is already transgenic. The most widely usedplant resistance markers are probably those for the antibiotickanamycin (see Kanamycin Selection of Transformed Arabidopsis)and the herbicide phosphinothricin or glufosinate ammonium,better known by its trade names Basta and Finale. An advantageof the latter is that it can be used for selection of transgenicplants on soil (see Glufosinate Ammonium Selection of Transformed Arabidopsis).

We use, for example, derivatives of pCGN1578 (McBride and Summerfelt 1990)and of the pPZP series (Hajdukiewicz et al. 1994). We have found,however, that the cauliflower mosaic virus (CaMV) 35S promoterdriving the resistance marker in the original pPZP vectors canlead to ectopic expression of the gene carried on the T-DNA,because the CaMV 35S promoter is right next to the multiplecloning site. This becomes a problem when predictable, tissue-specifictransgene expression is required. This problem can be solvedby replacing the promoter/resistance marker combination in pPZPwith one from a different vector.

Agrobacterium Strains

Many of the older protocols used strain LBA4404, but this strainoften does not appear to be virulent enough for the vacuum-infiltrationmethod. Better strains are C58 derivatives such as GV3101 (pMP90),GV3101 (pMP90RK) (Koncz and Schell 1986), and AGL-1 (Lazo et al. 1991).

REFERENCES

Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12: 8711–8721.

Hajdukiewicz P., Svab Z., Maliga P. 1994. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25: 989–994.

Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. 2000a. pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42: 819–832.

Hellens R, Mullineaux P, Klee H. 2000b. A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5: 446–451.

Jones J.D., Shlumukov L., Carland F., English J., Scofield S.R., Bishop G.J., Harrison K. 1992. Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res. 1: 285–297.

Koncz C. and Schell J. 1986. The promoter of the T_L-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383–396.

Lazo G.R., Stein P.A., Ludwig R.A. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technology 9: 963–967.

McBride K.E. and Summerfelt K.R. 1990. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 14: 269–276.

Table

Table 1. T-DNA vectors

Vector	Unique cloning sites	LacZ selection?	Resistance in		Reference
Vector	Unique cloning sites	LacZ selection?	bacteria	plants	Reference

pBIN19	9	no	kan	kan	Bevan(1984)
pCAMBIA series	variable	yes (not all)	chlor, kan	hyg,kan	http://www.cambia.org.au
pCGN series	5	yes	gent	kan	McBrideand Summerfelt (1990)
pJJ/pSLJ series	5-11	yes	tet	bar, kan,hyg, spec	Jones et al. (1992)
pPZP series	9	yes	chlor, spec	kan,gent	Hajdukiewicz et al. (1994)
pGreen series	18	yes	kan	bar,kan, hyg, sul	Hellens et al. (2000a)

Abbreviations for antibiotics:(bar) glufosinate ammonium (Basta), (chlor) chloramphenicol,(gent) gentamycin, (hyg) hygromycin, (kan) kanamycin, (sul)sulfonamide, (spec) spectinomycin, and (tet) tetracycline.

Rapid Characterization of DNAs Cloned in Prokaryotic Vectors

2008-04-24T12:53:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

In this method, sequences cloned in standard bacteriophage orplasmid vectors are amplified in PCRs containing primers targetedto flanking vector sequences. The amplified fragments can beanalyzed by gel electrophoresis, DNA sequencing, and/or restrictionmapping. Many colonies or plaques can be assayed simultaneously.To reduce the chance of contamination with exogenous DNAs, prepareand use a special set of reagents and solutions for PCR only.Bake all glassware for 6 hours at 150°C and autoclave allplasticware.

MATERIALS

10x Amplification buffer

Include 0.01% (w/v) gelatin in the buffer.

Forward primers (20 µM) in H₂O

Reverse primers (20 µM) in H₂O (20 pmoles/µl)

Template DNAs.

This method uses unpurified templates obtained by cracking bacteriophage particles or transformed bacterial cells. Essentially the samemethods can be used to analyze viral DNAs in productively infectedmammalian or yeast cells transformed by multicopy vectors.

Thermostable DNA polymerase

For advice on which enzyme to use, please see Table 12-19 inProtocol 5 in the print version of the manual. This protocolhas been written with AmpliTaq CS in mind, but it will workwell for thermostable enzymes with similar properties.

Tris-Cl (10 mM, pH 7.6)

dNTP solution (20 mM) containingall four dNTPs (pH 8.0)

METHOD

1. Calculate the number of bacterial colonies or bacteriophage plaques that are to be screened. Prepare the appropriate amountof master mix; analysis of each colony or plaque requires 25µl of master mix; 1 ml of master mix contains:

10x amplificationbuffer		100 µl
20 mM solution of four dNTPs		50 µl
forwardprimer		1 nmole
reverse primer		1 nmole
H₂O		to 1 ml

2.Dispense 25-µl aliquots of the master mix into theappropriatenumber of amplification tubes.

3. Use a sterile 200-µlpipette tip (NOT a toothpick)to touch each bacterial colonyor bacteriophage plaque. Workingquickly, wash the pipettetip in 25 µl of master mix.

4. Close the caps of thetubes. Incubate the closed tubes ina boiling water bath for10 minutes (bacterial colonies) or2 minutes (bacteriophage plaques).

5. Dilute the required amount of Taq DNA polymeraseto a concentrationof 1 unit/µl in 10 mM Tris (pH 7.6).Store the dilutedenzyme on ice.

6. Allow the samples (fromStep 4) to cool to room temperature.Centrifuge the tubes brieflyand then add 1 µl of thediluted Taq DNA polymerase toeach tube.

7. Set up two control reactions. In one reaction,include allof the components, except the template DNA. In theother reaction,include a recombinant bacteriophage plaqueor transformed bacteriallysate that has previously produceda positive result in thisassay.

8. If the thermal cyclerdoes not have a heated lid, overlaythe reaction mixtures with1 drop (approx. 50 µl) of lightmineral oil. Place thetubes in the thermal cycler. Amplifythe nucleic acids usingthe denaturation, annealing, and polymerizationtimes and temperatureslisted in the table.

Cycle Number		Denaturation		Annealing		Polymerization
30cycles		1 min at 94°C		2 min at 50°C		2 min at 72°C

Times and temperatures may need to be adapted to suit theparticularreaction conditions.

9. Withdraw a sample (5-10µl) from the test reactionmixture and the control reactionsand analyze them by electrophoresisthrough an agarose gel (Agarose Gel Electrophoresis).IncludeDNA markers of an appropriate size. Stain the gel withethidiumbromide or SYBR Gold to visualize the DNA (Detection of DNA in Agarose Gels).

A successful amplification reaction should yield a readilyvisibleDNA fragment. Nonrecombinant PCR products will be equalto thelength of DNA between the locations of the 5' terminiof thetwo primers in the cloning vector. Recombinant PCR productswill be the sum of (i) the length of the insert and (ii) thedistance between the 5' termini of two primers in the vector.If necessary, the identity of the band can be confirmed by restrictionmapping and Southern hybridization (Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes).

REFERENCES

1. Gussow, D. and Clackson, T. 1989. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17: 4000–4000.

2. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.

3. Sandhu, G.S., Precup, J.W., and Kline, B.C. 1993. Characterization of recombinant DNA vectors by polymerase chain reaction analysis of whole cells. Methods Enzymol. 218: 363–368.

Recipe

Amplification Buffer, 10X

500mM KCl

100 mM Tris-Cl (pH 8.3 atroom temperature)

15mM MgCl₂

Autoclave the 10x buffer for 10 minutes at 15 psi (1.05 kg/cm²) on liquid cycle. Divide the sterile buffer into aliquotsand store them at -20°C.

Recipe

Tris-Cl

Tris base

HCl

To prepare a 1 M solution, dissolve 121.1 g of Tris base in800 mL of H₂O. Adjust the pH to the desired value by addingconcentrated HCl.

pH	HCl

7.4	70 mL
7.6	60 mL
8.0	42mL

Allow the solution to cool to room temperature before makingfinal adjustments to the pH. Adjust the volume of the solutionto 1 L with H₂O. Dispense into aliquots and sterilize by autoclaving.

Recipe

dNTP solution

Dissolve each dNTP (deoxyribonucleoside triphosphates) in H₂Oat an approximate concentration of 100 mM. Use 0.05 M Tris baseand a micropipette to adjust the pH of each of the solutionsto 7.0 (use pH paper to check the pH). Dilute an aliquot ofthe neutralized dNTP appropriately, and read the optical densityat the wavelengths given in the table below. Calculate the actualconcentration of each dNTP. Dilute the solutions with H₂O toa final concentration of 50 mM dNTP. Store each separately at-70°C in small aliquots. For polymerase chain reactions(PCRs), adjust the dNTP solution to pH 8.0 with 2 N NaOH. Commerciallyavailable solutions of PCR-grade dNTPs require no adjustment.

Base	wavelength (nm)	Extinction Coefficient (E) (M^-1cm^-1)
A	259	1.54x 10⁴
G	253	1.37 x 10⁴
C	271	9.10 x 10³
T	267	9.60 x 10³

For a cuvette with a path length of 1 cm, absorbance = EM. 100mM stock solutions of each dNTP are commercially available (Pharmacia).

Cloning PCR Products into T Vectors

2008-04-24T12:50:00.001-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

This method of direct cloning takes advantage of the unpairedadenosyl residue added to the 3' terminus of amplified DNAsby Taq and other thermostable polymerases.

MATERIALS

Bacteriophage T4 DNA ligase

T vector

Target DNA (25 µg/ml), amplified by PCR

When the PCR mixture contains more than one or two bands ofamplified DNA, purify the target fragment by electrophoresisthrough low melting/gelling temperature agarose (please seeRecovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction).If not purified by gel electrophoresis, PCR-amplified DNA shouldbe prepared for ligation by extraction with phenol:chloroformand ultrafiltration through a Centricon-100 filter (please seeRemoval of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration).

METHOD

1. In a microcentrifuge tube, set up the following ligationmixture:

25 µg/ml amplified target DNA		1 µl
T-tailedplasmid		20 ng
10x ligation buffer		1 µl
bacteriophageT4 DNA ligase		3 units
H₂O		to 10 µl

If necessary,add ATP to a final concentration of 1 mM. A 1:5molar ratioof vector:amplified DNA fragment is recommended.

Set upa control reaction that contains all the reagents listedaboveexcept the amplified target DNA.

2. Incubate the ligationmixture for 4 hours at 14°C.

3. Dilute 5 µl of eachof the two ligation mixtures with10 µl of H₂O and transforma suitable strain of competentE. coli to antibiotic resistanceas described in Preparation and Transformation of Competent E. coli Using Calcium ChlorideorTransformation of E. coli by Electroporation. Plate the transformedcultures on media containing IPTG and X-gal (please see Screening Bacterial Colonies Using X-gal and IPTG: -Complementation)and the appropriate antibiotic.

4. Calculate the number ofcolonies obtained from each of theligation mixtures. Pick anumber of white colonies obtainedby transformation with theligation reaction containing thetarget DNA. Confirm the presenceof the amplified fragment by(i) isolating the plasmid DNAsand digesting them with restrictionenzymes whose sites flankthe insert in the multiple cloningsite or (ii) colony PCR (Rapid Characterization of DNAs Cloned in Prokaryotic Vectors.

The ratio of blue:white colonies varies between 1:5 and 2:1.

5. Fractionate the restricted DNA by electrophoresis throughan agarose gel using appropriate DNA size markers. Measure thesize of the cloned fragments.

6. Confirm the identity of thecloned fragments by DNA sequencing(Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers),restriction mapping, or Southern hybridization (Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes).

REFERENCES

1. Holton, T.A. and Graham, M.W. 1991. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19: 1154–1154.

2. Marchuk, D., Drumm, M., Saulino, A., and Collins, F.S. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1156–1156.

Cloning PCR Products into T Vectors

2008-04-24T12:50:00.000-07:00

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

This method of direct cloning takes advantage of the unpairedadenosyl residue added to the 3' terminus of amplified DNAsby Taq and other thermostable polymerases.

MATERIALS

Bacteriophage T4 DNA ligase

T vector

Target DNA (25 µg/ml), amplified by PCR

METHOD

1. In a microcentrifuge tube, set up the following ligationmixture:

25 µg/ml amplified target DNA		1 µl
T-tailedplasmid		20 ng
10x ligation buffer		1 µl
bacteriophageT4 DNA ligase		3 units
H₂O		to 10 µl

If necessary,add ATP to a final concentration of 1 mM. A 1:5molar ratioof vector:amplified DNA fragment is recommended.

Set upa control reaction that contains all the reagents listedaboveexcept the amplified target DNA.

2. Incubate the ligationmixture for 4 hours at 14°C.

The ratio of blue:white colonies varies between 1:5 and 2:1.

5. Fractionate the restricted DNA by electrophoresis throughan agarose gel using appropriate DNA size markers. Measure thesize of the cloned fragments.

REFERENCES

1. Holton, T.A. and Graham, M.W. 1991. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19: 1154–1154.

Transduction of Cell Lines by Retroviral Vectors

2008-04-24T12:49:00.000-07:00

Kenneth Cornetta, Karen E. Pollok, and A. Dusty Miller

This protocol was adapted from "Retroviral Vectors," Chapter 2, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

This protocol is suitable for transduction of many adherentcell lines. The number of target cells transduced can be variedas needed by maintaining the ratio of surface area to volumeand using plates/flasks of various sizes. The protocol can alsoeasily be adapted for non-adherent cells using similar vector-to-cellratios.

RELATED INFORMATION

This issue of CSH Protocols contains the related articles Retroviral Vector Production by Transient Transfectionand Generation of Stable Vector-Producing Cells for Retroviral Vectors.

MATERIALS

Reagents

Cell culture medium (normal growth medium for the cells beingtransduced)

Drug for selection (optional; see Step 7.i)

Vectors are frequently generated that express the specific transgeneof interest along with a "marker" gene that allows for enrichmentof transduced populations.

Protamine sulfate or Polybrene (see Step 4)

A stock solution of 1000X polycation can be made for convenience.

Retroviral vector stock

See Retroviral Vector Production by Transient Transfection orGeneration of Stable Vector-Producing Cells for Retroviral Vectors.

Target cells

Equipment

Aspirator

Cell sorter (optional; see Step 7.ii)

Incubator, preset to 37ºC, 5% CO₂

Pipettes (sterile, disposable)

Tissue-culture flasks (75 cm²)

Water bath, preset to 37ºC

METHOD

Prepare for Transduction

1. Prepare cultures for transduction. Include a mock-transductioncontrol.
If drug selection of the transduced population isanticipated,prepare a second mock-transduced control that willserve asa drug selection control to document drug potency.

i. Prepare a single-cell suspension of target cells, and calculatethe number of cells per milliliter.

ii. For adherent cells,plate 5 x 10² cells in each 75-cm² tissue-cultureflask with12 mL of appropriate medium.

iii. Incubate overnight in a37ºC, 5% CO₂ incubator.

2. Rapidly thaw the vectorin a 37ºC water bath and preparedilutions, if appropriate.Minimize the time between thawingand cell exposure.

3. Removemedium from the cells by aspiration and discard.

Perform the Transduction

4. Add the vector to the cells as follows:

i. Vector transduction:Add 4 mL of vector-containing mediumsupplemented with 8 µg/mLPolybrene or 10 µg/mLprotamine sulfate to each 75-cm²flask.

ii. Mock transduction:Add 4 mL of fresh medium supplementedwith 4 µg/mL Polybreneor 10 µg/mL protamine sulfateto the appropriate controlcultures.
Polycations such as Polybreneor protamine sulfateare addedto increase the interaction ofthe negatively chargedcellsand vector particles and can increasetransduction 10-foldormore. Polycations can be toxic to primarycells and somecelllines. Test a variety of lower concentrationsto identifythedose with minimal toxicity and optimal genetransfer.

5. Return cells to a 37ºC, 5% CO₂ incubator.Expose cellsto vector for 4 h.
With cell lines, incubationover 4 h generally does not increasetransduction efficiencysignificantly. To increase gene transfer,it is preferable torepeat the transduction procedure on thesubsequent day(s),presumably when additional cells have enteredinto a favorablepart of the cell cycle for transduction.

6. After the incubation,aspirate the medium from the flasks,add fresh medium, and returnthe cells to incubator.
Stable expression of integrated transgeneis seen ~72 h aftertransduction of most cell lines.
See Troubleshooting.

7. For drug selection or cell sorting (optional):

i. Ifusing drug selection, add the drug 24-48 h after transduction.

ii. In cases where surface expression molecules or fluorescentproteins are expressed that facilitate sorting of vector-expressingcells, wait 72 h to allow adequate time for vector expressionin the majority of transduced cells.

8. Cells can be usedfor analysis or expanded for subsequentuse.

TROUBLESHOOTING

Problem: Gene-transfer efficiency is low.

[Step 6]

Solution: Three factors should be considered: multiplicity ofinfection (MOI), vector titer, and cell density at the timeof transduction. The optimal MOI varies with each cell line.For most immortalized cell lines, an MOI of 10-20 infectiousvector particles per each cell is adequate, especially if selectionof transduced cells is planned. Primary cells usually requirehigher MOI (when possible), colocalization, or multiple roundsof transduction. Above the optimal MOI, the transduction efficiencytends to plateau, although the number of integrations per cellcan increase. The vector titer (or concentration) is also afactor, and generally, the more concentrated the material, thehigher the level of gene transfer (up to the plateau level).

Generation of Stable Vector-Producing Cells for Retroviral Vectors

2008-04-24T12:43:00.001-07:00

Kenneth Cornetta, Karen E. Pollok, and A. Dusty Miller

INTRODUCTION

This procedure describes the generation of clonal vector-producingcells that will provide an unlimited amount of unrearrangedretroviral vector. The procedure involves transfection of onepackaging cell line to generate a vector that is used to transducea second packaging cell line. The resultant vector-producingclones generally contain a single integrated copy of the retroviralvector, and virus produced from this integrated vector is asgenetically homogeneous as possible. Although the vector producedby a given packaging cell line can sometimes be used to transducethe same cell line, the transduction rate is typically low becauseof receptor blockage by the Env protein made by the target packagingcells. Indeed, this procedure will select for target cells thatexpress low Env protein levels and thus are less resistant totransduction, but at the same time will ultimately produce lessvector because of low Env production. Therefore, to obtain thehighest vector titers, it is important to use pairs of packagingcells such that receptor blockage is not an issue. In this example,we use PE501 ecotropic packaging cells for transfection andbroad-host-range PT67 packaging cells to make stable vector-producingcells.

RELATED INFORMATION

This issue of CSH Protocols contains the related article Retroviral Vectors for Gene Transfer,which provides an overview of issues to consider when designinggene-transfer experiments involving retrovirus vectors. Theretrovirus vector produced in this protocol may be subsequentlyused for transduction (see Transduction of Cell Lines by Retroviral Vectorsand Transduction of Primary Hematopoietic Cells by Retroviral Vectors,also this issue).

MATERIALS

Reagents

Sterilize all transfection reagents before use by filtrationthrough 0.22-µm, pore-size sterile filters.

CaCl₂ (2.0 M)

Chemical for selection

Depending on the selectable marker in the vector, choices include:

G418

Histidinol

HygromycinB

Dulbecco’s modified Eagle’s medium (DMEM) with 10%fetal bovine serum (FBS)

Dye for staining cells

Packaging cells

Suitable combinations of packaging cell lines are shown formouse (Table 1) and human (Table 2). Differences in these tablesrelate to the inability of ecotropic vectors to transduce humancells and the inability of gibbon ape leukemia virus (GALV)and RD114 vectors to transduce mouse cells. In this protocol,we use PE501 ecotropic packaging cells for transfection andbroad-host-range PT67 packaging cells to make stable vector-producingcells.

Polybrene (4 mg/mL) in phosphate-buffered saline (PBS), sterile-filtered(Sigma-Aldrich)

This is a 1000X stock solution of polybrene; for use, diluteto 4 µg/mL in appropriate medium.

Precipitation buffer

Recipient cells (e.g., NIH-3T3 or HeLa)

Trypsin-EDTA

Vector plasmid (must carry selectable marker) in 10 mM Tris-Cl(pH 7.5)

Include a vector that carries only the selectable marker geneand not the desired cDNA insert as a positive control to monitorsuccess of the various steps of the procedure. Use a plasmidthat does not carry a retroviral vector as a negative control.Prepare endotoxin-free vector plasmid stocks (e.g., by usingthe QIAGEN Endotoxin-free Purification Kit) and determine theplasmid DNA concentration.

Equipment

Aspirator

Biosafety cabinet

Centrifuge

Cloning rings

CO₂ incubator

Felt-tip pen

Microscope

Petri dishes (10 cm), glass

Pipettes (sterile, disposable)

Silicone grease (Dow Corning high-vacuum grease or equivalent)

Tissue-culture dishes (6 and 10 cm)

Tube (clear polystyrene, 12 x 75 mm) (Falcon 2054 or equivalent)

Tweezers

METHOD

Preparation of Clonal Lines

Figure 1 outlines this procedure.

Figure 1. Generation of retroviral packaging cell lines.

1. Day 1: Seed PE501 ecotropic retroviral packaging cells at5 x 10⁵ cells per 6-cm dish and incubate overnight.

2. Day2: Replace the culture medium on the PE501 cells with4 mL offresh medium, and transfect the cells with vector plasmidDNAusing the calcium phosphate precipitation procedure as follows:

i. For each plasmid sample, prepare a DNA-CaCl₂ solution bymixing the following together:

25 µL of 2.0 M CaCl₂
10 µg of plasmid DNA (in10 mM Tris-Cl at pH 7.5)
H₂Oto make 200 µL total

ii. Prepare fresh precipitationbuffer. In a clear 12 x 75-mmpolystyrene tube, add 200 µLof DNA-CaCl₂ solution drop-wiseto 200 µL of precipitationbuffer with constant agitation.
A faint cloudiness in thesolution should be immediately apparent.If the mixture remainsclear or a precipitate consisting oflarge clumps develops,something is wrong.

iii. After 30 min at room temperature,add the resultant fineprecipitate to a dish of cells, and swirlthe dish to distributethe precipitate. Incubate overnight.

3. Day 3: Aspirate the medium from the transfected PE501cells,and add 4 mL of fresh medium. Seed PT67 packaging cellsat 10⁵cells per 6-cm dish, two dishes for each dish of transfectedPE501 cells. Incubate overnight.

4. Day 4: Infect cells asfollows:

i. Replace the medium on the PT67 cells with mediumcontaining4 µg/mL Polybrene.

ii. Remove 3 mL of virus-containingmedium from each dish oftransfected PE501 cells (retain 1 mLto keep the cells fromdrying out until they are trypsinized).

iii. Centrifuge the medium at 3000g for 5 min to remove cellsand debris.

iv. From each dish of transfected PE501 cells,use 1 mL of virus-containingmedium to infect one dish of PT67cells, and 10 µL toinfect another dish of PT67 cells.Return the PT67 cells tothe incubator.

5. Trypsinize andseed the PE501 cells at a 1:20 dilution into6-cm dishes containingmedium with 0.75 mg/mL G418 (active concentration),4 mM histidinol,or 0.4 mg/mL hygromycin B, depending on theselectable markerin the vector. Return the cells to the incubator.

6. Day 5:Trypsinize the infected PT67 cells, and seed the cellsat 9:10and 1:10 dilutions into 10-cm dishes containing 10 mLof mediumplus the appropriate drug for selection (see Step5).
The9:10 and 1:10 dilutions of PT67 cells infected with 1 mLor10 µL of virus result in a 4-log range of dilutions,someof which should yield appropriate numbers of colonies forisolationof clonal cell lines.

7. Day 9: Stain the PE501 cells andevaluate for colony formationafter 5 d of selection as a measureof the efficiency of DNAtransfection. A transfection efficiencyof about 1000 coloniesper microgram of plasmid DNA is typical.

8. Days 9-14: Passage the transduced PT67 cells for 5-10 d,until drug-resistant colonies form and are clearly visible.

9. Use cloning rings to isolate clones from dishes containingsmall numbers of colonies.

i. To prepare the cloning ringsfor use, spread a thin coatingof silicone grease on the bottomplate of a 10-cm glass Petridish. Place the rings in the dishso that the grease coats oneopen end, and autoclave the dishto sterilize.

ii. To isolateclones, locate colonies and drawa circle aroundeach colonyon the bottom of the dish with afelt-tip pen.
Colonies canbe most easily visualized by holdingthe dish upto the light,taking care not to spill the medium.Turn offthe airflow inthe laminar airflow hood to avoid desiccationof the coloniesduring placement of cloning rings.

iii. Aspiratethe medium,place cloning rings over coloniesto be isolated,and pressdown with tweezers.

10. Add a drop of trypsin-EDTAto each cylinder, and monitorthe extent of trypsinization microscopically.

11. When the cells have rounded up, add medium to each ring(one at a time) and, fairly vigorously, force the medium inand out of a pipette to dislodge the cells.
We typically isolateapproximately 10 colonies for analysis.

12. After expansion,assay the clonal lines for an intact vectorstructure by Southernanalysis, for the production of high vectortiter, for the presenceof helper virus (see Marker Rescue Assay,Steps 15-24), andfor expression of the inserted gene.

Virus Harvest and Assay

13. To prepare virus, replace the medium on confluent culturesof vector-producing cells. Collect the medium 12-24 h later,and centrifuge the medium at 3000g for 5 min to remove cellsand debris.
This process can be repeated three or four timesat 12-h intervalsfrom the same dish of cells. The virus-containingmedium canbe used immediately to infect recipient cells orfrozen at -70°Cfor later use.

14. Determine vector titeras follows:

i. Seed recipient cells (e.g., NIH-3T3 or HeLa)at 5 x 10⁵ per6-cm dish and grow overnight.

ii. Change themedium to mediumcontaining 4 µg/mL Polybreneand addvarious dilutionsof test virus. Incubate overnight.

iii.Trypsinize and dilutethe cells 1:20 into medium containing0.75 mg/mL G418 (activeconcentration) for vectors carryingthe neo gene, 4 mM histidinolfor vectors carrying the hisDgene, and 0.4 mg/mL hygromycinB for vectors carrying the hphgene. Adjust these concentrationsdepending on the cell line.Return to incubator.

iv. After5-7 d, stain and count colonies.Calculate virus titerin colony-formingunits per milliliter(CFU/mL) by dividingthe number of coloniesby the volume (inmilliliters) of virusused for infection andmultiplying by20 to correct for the1:20 cell dilution.

Marker Rescue Assay for Helper Virus

This assay measures the ability of a viral sample to rescueor mobilize a retroviral vector from cells that contain butdo not produce a vector. The assay depends on the ability ofthe helper virus to infect the cells used. For example, ecotropichelper virus cannot be detected by using human cells. Assaycells should be chosen to match the expected helper viruses.This assay is somewhat slow, but it is very sensitive and measuresthe most important property of helper viruses in the contextof retroviral vector design, the ability to mobilize vectors.

15. To make cells that harbor a vector but do not release avector, infect NIH-3T3 or HeLa cells with a helper-free vectorcarrying a selectable marker (we use the LN vector that carriesthe neo gene) and select the cells for the presence of the selectablegene (G418 for neo).
This virus can be obtained from a packagingline that producesany vector at high titer.

16. Passage thecells for 2 wk to allow potential helper virus(which shouldnot be present) to spread. Assay the cells forvector productionby using NIH-3T3 or HeLa cells as indicatorcells for virusproduction.

17. Preserve cells that do not produce the vector(nonproducercells) for use in the marker rescue assay describedbeginningwith Step 4.

18. Seed nonproducer cells containinga neo vector (NIH-3T3or HeLa cells) identified in Step 3 at5 x 10⁵ per 6-cm dishand grow overnight.

19. Infect nonproducercells by adding 1 mL of test virus (centrifugedat 3000g for5 min to remove cells and debris), 3 mL of regularmedium, and4 µg/mL Polybrene. Infect control positivedishes witha small amount of amphotropic helper virus plasmid(e.g., 1µL or less of virus produced by NIH-3T3 cellstransfectedwith pAM-MLV and passaged for 2 wk to allow completeinfectionof the cells) or other helper virus capable of replicatinginthe nonproducer cells.

20. Passage cells for 2 wk to allowhelper virus spread (3 wkif attempting to qualify clinicalgrade material). Take carenot to cross-contaminate the cultures,some of which may beginto make helper virus at very high titer.Trypsinize the cellstwo to three times a week, and replatethe cells at 1:10 to1:40 dilutions. Keep the cells at relativelyhigh density tofacilitate viral spread.

21. After 13 or 20d of passaging nonproducer cells (see Step20), plate naiveNIH-3T3 or HeLa cells (same cell type as nonproducercell lineused) at 10⁵ per 6-cm dish. Grow overnight. Feed confluentdishesof nonproducer cells (which now may be "producing" virus).

22.Harvest medium from the nonproducer cells, and use 1-mLsamplesto infect the naive NIH-3T3 or HeLa cells in the presenceof4 µg/mL Polybrene. Centrifuge the medium at 3000g for5 min to remove cells and debris. Any live cells that are transferredalong with the medium will be drug-resistant and could givea false-positive result. Return the newly infected cells tothe incubator and grow overnight.

23. Replace the medium onthe newly infected cells with mediumcontaining G418 (0.75 mg/mLactive concentration for NIH-3T3or 1.0 mg/mL active concentrationfor HeLa).

24. After 5 d, stain and count colonies.
Thepresence of colonies indicates that the neo vector was rescuedby helper virus in the test sample. Usually this is very obvious,and positive dishes are covered with drug-resistant colonies.

Caution

CaCl₂ (calcium chloride)

CaCl₂ (calcium chloride) is hygroscopic and may cause cardiacdisturbances. It may be harmful by inhalation, ingestion, orskin absorption. Do not breathe the dust. Wear appropriate glovesand safety goggles.

Caution

G418 (an aminoglycosidic antibiotic)

G418 (an aminoglycosidic antibiotic) is toxic and may causeharm to the unborn child. It may be harmful by inhalation, ingestion,or skin absorption. Wear appropriate gloves and safety gogglesand use in a chemical fume hood. Do not breathe the dust.

Caution

Hygromycin B

Hygromycin B is highly toxic and may be fatal if inhaled, ingested,or absorbed through the skin. Wear appropriate gloves and safetygoggles. Use only in a chemical fume hood. Do not breathe thedust.

Recipe

Precipitation buffer

Reagent	Quantity

HEPES-NaOH (500 mM, pH 7.1)	100 µL
NaCl(2.0 M)	125 µL
Na₂HPO₄-NaH₂PO₄ (150 mM, pH 7.0)	10 µL
H₂O	to1 mL (final volume)
Prepare fresh.

Recipe

Trypsin-EDTA

0.05% trypsin

0.5 mM EDTA (pH 8.0)

Table

Table 1. Susceptibility of mouse packaging cell lines to transduction with vectors produced by other packaging cell lines

		Susceptibility of target mouse packaging cells
Vector-producingpackaging cells	Virus receptor	Ecotropic	Amphotropic	10A1	GALV	RD114

Ecotropic	CAT-1	-	+	+	+	+
Amphotropic	Pit2	+	-	-	+	+
10A1	Pit1or Pit2	+	+	-	+	+
GALV	Pit1	-	-	-	-	-
RD114	RDR	-	-	-	-	-

Table

Table 2. Susceptibility of human packaging cell lines to transduction with vectors produced by other packaging cell lines

		Susceptibility of target human packaging cells
Vector-producingpackaging cells	Virus receptor	Ecotropic	Amphotropic	10A1	GALV	RD114

Ecotropic	CAT-1	-	-	-	-	-
Amphotropic	Pit2	+	-	-	+	+
10A1	Pit1or Pit2	+	+	-	+	+
GALV	Pit1	+	+	-	-	+
RD114	RDR	+	+	+	+	-

Generation of Stable Vector-Producing Cells for Retroviral Vectors

2008-04-24T12:43:00.000-07:00

Kenneth Cornetta, Karen E. Pollok, and A. Dusty Miller

INTRODUCTION

RELATED INFORMATION

MATERIALS

Reagents

Sterilize all transfection reagents before use by filtrationthrough 0.22-µm, pore-size sterile filters.

CaCl₂ (2.0 M)

Chemical for selection

Depending on the selectable marker in the vector, choices include:

G418

Histidinol

HygromycinB

Dulbecco’s modified Eagle’s medium (DMEM) with 10%fetal bovine serum (FBS)

Dye for staining cells

Packaging cells

Polybrene (4 mg/mL) in phosphate-buffered saline (PBS), sterile-filtered(Sigma-Aldrich)

This is a 1000X stock solution of polybrene; for use, diluteto 4 µg/mL in appropriate medium.

Precipitation buffer

Recipient cells (e.g., NIH-3T3 or HeLa)

Trypsin-EDTA

Vector plasmid (must carry selectable marker) in 10 mM Tris-Cl(pH 7.5)

Equipment

Aspirator

Biosafety cabinet

Centrifuge

Cloning rings

CO₂ incubator

Felt-tip pen

Microscope

Petri dishes (10 cm), glass

Pipettes (sterile, disposable)

Silicone grease (Dow Corning high-vacuum grease or equivalent)

Tissue-culture dishes (6 and 10 cm)

Tube (clear polystyrene, 12 x 75 mm) (Falcon 2054 or equivalent)

Tweezers

METHOD

Preparation of Clonal Lines

Figure 1 outlines this procedure.

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Figure 1. Generation of retroviral packaging cell lines.

1. Day 1: Seed PE501 ecotropic retroviral packaging cells at5 x 10⁵ cells per 6-cm dish and incubate overnight.

2. Day2: Replace the culture medium on the PE501 cells with4 mL offresh medium, and transfect the cells with vector plasmidDNAusing the calcium phosphate precipitation procedure as follows:

i. For each plasmid sample, prepare a DNA-CaCl₂ solution bymixing the following together:

25 µL of 2.0 M CaCl₂
10 µg of plasmid DNA (in10 mM Tris-Cl at pH 7.5)
H₂Oto make 200 µL total