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Chapter 3 Working with bacteriophage M13 Vectors

Chapter 3 Working with Bacteriophage M13 Vectors

Protocol 1: Plating Bacteriophage M13

Bacteriophage M13 forms turbid plaques on lawns of male strains of E. coli.

Protocol 2: Growing Bacteriophage M13 in Liquid Culture

Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate.

Protocol 3: Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA

The double-stranded replicative form (RF) of bacteriophage M13 is isolated from infected cells using methods similar to those used to purify plasmid DNA. Several micrograms of RF DNA can be isolated from a 1-2-ml culture of infected cells.

Protocol 4: Preparation of Single-stranded Bacteriophage M13 DNA

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. Typically, the yield of single-stranded DNA

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Chapter 3 Working with bacteriophage M13 Vectors

is 5-10 µg/ml infected culture.

Protocol 5: Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA

This protocol, a scaled-up version of Chapter 3, Protocol 3 and Chapter 3, Protocol 4 , is used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants.

Protocol 6: Cloning into Bacteriophage M13 Vectors

This protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning.

Protocol 7: Analysis of Recombinant Bacteriophage M13 Clones

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants.

Protocol 8: Producing Single-stranded DNA with Phagemid Vectors

This protocol describes methods to superinfect bacteria carrying a recombinant phagemid with a high-titer stock of an appropriate helper virus and to assay the yield of filamentous virus particles that carry single-stranded copies of the phagemid DNA. The key to success in using phagemids is to prepare a stock of helper virus whose titer is accurately known.

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Chapter 3 Working with bacteriophage M13 Vectors

Chapter 3, Protocol 1

Plating Bacteriophage M13

Bacteriophage M13 forms turbid plaques on lawns of male strains of E. coli. CAUTION RECIPE

MATERIALS

Buffers and Solutions IPTG (20% w/v)

X-gal solution (2% w/v)

Media

LB agar plates containing tetracycline or kanamycin

These plates are needed only if a tetracycline-resistant strain of E. coli, such as XL1-Blue, or a kanamycin-resistant strain of E. coli, such as XL1-Blue MRF´ Kan, is used to propagate the virus.

M9 minimal agar plates, supplemented biosynthetic operon (Rich M13 medium

These plates are needed when using E. coli strains that carry a deletion of the proline

[lac-proAB]) in the bacterial chromosome and the complementing

proAB genes on the F´ plasmid.

Rich M13 top agar or agarose containing 5 mM MgCl2

Vectors and Bacterial Strains Bacteriophage M13 stock

Rich M13 agar medium plates containing 5 mM MgCl2

LB or YT medium from a fully grown liquid culture of bacteria infected with bacteriophage M13 contains between 10 and 10 pfu/ml. A bacteriophage M13 plaque contains between 10 and 10 pfu.

E. coli F´ strain, prepared as a master culture

METHOD

1. Streak a master culture of a bacterial strain carrying an F´ plasmid onto either a

supplemented minimal (M9) agar plate or an LB plate containing tetracycline

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Chapter 3 Working with bacteriophage M13 Vectors

(XL1-Blue) or kanamycin (XL1-Blue MRF´ Kan). Incubate the plate for 24-36 hours at 37°C.

2. To prepare plating bacteria, inoculate 5 ml of LB or YT medium in a 20-ml sterile

culture tube with a single, well-isolated colony picked from the agar plate prepared in Step 1. Agitate the culture for 6-8 hours at 37°C in a rotary shaker. Chill the culture in an ice bath for 20 minutes and then store it at 4°C. These plating bacteria can be stored for periods of up to 1 week at 4°C.

Do not grow the cells to saturation, as this will increase the risk of losing the pili encoded by the F´ plasmid.

3. Prepare sterile tubes (13 x 100 mm or 17 x 100 mm) containing 3 ml of melted LB or

YT medium top agar or agarose, supplemented with 5 mM MgCl2. Allow the tubes to equilibrate to 47°C in a heating block or water bath.

4. Label a series of sterile tubes (13 x 100 mm or 17 x 100 mm) according to the dilution

factor and amount of bacteriophage stock to be added (please see Step 5), and deliver 100 µl of plating bacteria from Step 2 into each of these tubes.

5. Prepare tenfold serial dilutions (10 to 10) of the bacteriophage stock in LB or YT

medium. Dispense 10 µl or 100 µl of each dilution to be assayed into a sterile tube containing plating bacteria from Step 4. Mix the bacteriophage particles with the bacterial culture by vortexing gently.

6. Add 40 µl of 2% X-gal solution and 4 µl of 20% IPTG solution to each of the tubes

containing top agar. Immediately pour the contents of one of these tubes into one of the infected cultures. Mix the culture with the agar/agarose by gently vortexing for 3 seconds, and then pour the mixture onto a labeled plate containing LB or YT agar medium supplemented with 5 mM MgCl2 and equilibrated to room temperature. Swirl the plate gently to ensure an even distribution of bacteria and top agar.

Work quickly so that the top agar spreads over the entire surface of the agar before it sets.

7. Repeat the addition of top agar with X-gal and IPTG for each tube of infected culture

prepared in Step 5.

8. Replace the lids on the plates and allow the top agar/agarose to harden for 5 minutes

at room temperature. Wipe excess condensation off the lids with Kimwipes. Invert the plates and incubate them at 37°C.

Pale blue plaques begin to appear after 4 hours. The color gradually intensifies as the plaques enlarge and is complete after 8-12 hours of incubation.

-6

-9

RECIPES

5x M9 Salts Na2HPO4•7H2O, g KH2PO4, 15 g NaCl, 2.5 g NH4Cl, 5.0 g

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Chapter 3 Working with bacteriophage M13 Vectors

deionized H2O, to 1 liter

Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes

at 15 psi (1.05 kg/cm ) on liquid cycle.

CaCl2

Dissolve 11 g of CaCl2•6H2O in a final volume of 20 ml of distilled H2O. Sterilize the 2.5

M solution by passing it through a 0.22-µm filter. Store in 1-ml aliquots at 4°C.

IPTG

IPTG is isopropylthio--D-galactoside. Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled H2O. Adjust the volume of the solution to 10 ml

with H2O and sterilize by passing it through a 0.22-µm disposable filter. Dispense the solution into 1-ml aliquots and store them at -20°C.

deionized H2O, to 950 ml tryptone, 10 g yeast extract, 5 g NaCl, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1

2on liquid cycle.

liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm )

sterile H2O (cooled to 50°C or less), to 750 ml 5x M9 salts, 200 ml 1 M MgSO4, 2 ml

20% solution of the appropriate carbon source (e.g., 20% glucose), 20 ml 1 M CaCl2, 0.1 ml

sterile deionized H2O, to 980 ml

For solid medium, please see Media Containing Agar or Agarose If necessary, supplement the M9 minimal medium with stock solutions of the

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appropriate amino acids and vitamins.

Prepare the MgSO4 and CaCl2 solutions separately, sterilize by autoclaving, and add the solutions after diluting the 5x M9 salts to 980 ml with sterile H2O. Sterilize the glucose by passing it through a 0.22-µm filter before it is added to the diluted M9 salts. When using E. coli strains that carry a deletion of the proline biosynthetic operon [(lac-proAB)] in the bacterial chromosome and the complementing proAB genes on the F' plasmid, supplement the M9 minimal medium with the following: 0.4% (w/v) glucose (dextrose) 5 mM MgSO4•7H2O 0.01% thiamine

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following: Bacto Agar (for plates) agarose (for plates)

15 g/liter 15 g/liter

Bacto Agar (for top agar) 7 g/liter agarose (for top agarose) 7 g/liter

Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle. When the medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Allow the medium to cool to 50-60°C before adding thermolabile substances (e.g., antibiotics). To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored markers.

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Chapter 3 Working with bacteriophage M13 Vectors

This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37°C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.

MgSO4

To prepare a 1 M solution: Dissolve 12 g of MgSO4 in a final volume of 100 ml of H2O.

Sterilize by autoclaving or filter sterilization. Store at room temperature.

NaCl

to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl

solution at room temperature.

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume

Rich M13 LB YT

For solid medium, please see Media Containing Agar or Agarose.

X-gal Solution

X-gal is 5-bromo-4-chloro-3-indolyl--D-galactoside. Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use prevent damage by light and store at -20°C. It is not necessary to sterilize X-gal solutions by filtration.

a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g NaCl, 5 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0

with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize

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Chapter 3 Working with bacteriophage M13 Vectors

by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle.

CAUTIONS

X-gal

powder. Note that stock solutions of X-gal are prepared in DMF, an organic solvent. For

details, see DMF. See also BCIG.

X-gal may be toxic to the eyes and skin. Observe general cautions when handling the

Chapter 3, Protocol 2

Growing Bacteriophage M13 in Liquid Culture

MATERIALS

Media

LB containing tetracycline or kanamycin

These media are needed only if a tetracycline-resistant strain of E. coli, such as XL1-Blue, or a kanamycin-resistant strain of E. coli is used to propagate the virus. M9 minimal medium, supplemented

This media is needed when using E. coli strains that carry a deletion of the proline biosynthetic operon ([lac-proAB]) in the bacterial chromosome and the complementing proAB genes on the F´ plasmid. Rich M13 medium

2x YT medium containing 5 mM MgCl2

Vectors and Bacterial Strains

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Bacteriophage M13 plaques plated onto an agar or agarose plate Please see either Chapter 3, Protocol 1 or Chapter 3, Protocol 6 . E. coli F´ strain, grown as well-isolated colonies on an agar plate

METHOD

1. Inoculate 5 ml of supplemented M9 medium (or, for antibiotic-resistant strains, LB

medium with the appropriate antibiotic) with a single freshly grown colony of E. coli carrying an F´ plasmid. Incubate the culture for 12 hours at 37°C with moderate shaking.

2. Transfer 0.1 ml of the E. coli culture into 5 ml of 2x YT medium containing 5 mM MgCl2.

Incubate the culture for 2 hours at 37°C with vigorous shaking.

3. Dilute the 5-ml culture into 45 ml of 2x YT containing 5 mM MgCl2 and dispense 1-ml

aliquots into as many sterile tubes (13 x 100 mm or 17 x 100 mm) as there are plaques to be propagated. Dispense two additional aliquots for use as positive and negative controls for bacteriophage growth. Set these cultures aside for use at Step 7. 4. Dispense 1 ml of YT or LB medium into sterile 13 x 100-mm tubes. Prepare as many

tubes as there are plaques. Dispense two additional aliquots for use as positive and negative controls for bacteriophage growth.

5. Prepare a dilute suspension of bacteriophage M13 by touching the surface of a plaque

with the end of a sterile inoculating needle and immersing the end of the needle into the YT or LB medium. Pick one blue M13 plaque as a positive control for bacteriophage growth. Also pick an area of the E. coli lawn from the plate that does not contain a plaque as a negative control.

6. Allow the suspension to stand for 1-2 hours at room temperature to allow the

bacteriophage particles to diffuse from the agar.

7. Use 0.1 ml of the bacteriophage suspension (Step 6) as an inoculum to infect 1-ml

cultures of E. coli (Step 3) for isolation of viral DNA. Incubate the inoculated tubes for 5 hours at 37°C with moderate shaking.

Alternatively, transfer a plaque directly into the E. coli culture.

To minimize the possibility of selecting deletion mutants, grow cultures infected with recombinant M13 bacteriophages for the shortest time that will produce a workable amount of single-stranded DNA (usually 5 hours).

8. Transfer the culture to a sterile microfuge tube and centrifuge at maximum speed for 5

minutes at room temperature. Transfer the supernatant to a fresh microfuge tube without disturbing the bacterial pellet.

9. Transfer 0.1 ml of the supernatant to a sterile microfuge tube.

10. Use the remaining 1 ml of the culture supernatant to prepare single-stranded

bacteriophage DNA ( Chapter 3, Protocol 4 ). Use the bacterial cell pellet to prepare double-stranded RF DNA ( Chapter 3, Protocol 3 ).

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Chapter 3 Working with bacteriophage M13 Vectors

RECIPES

5x M9 Salts Na2HPO4•7H2O, g KH2PO4, 15 g NaCl, 2.5 g NH4Cl, 5.0 g

deionized H2O, to 1 liter

Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes

at 15 psi (1.05 kg/cm ) on liquid cycle.

CaCl2

Dissolve 11 g of CaCl2•6H2O in a final volume of 20 ml of distilled H2O. Sterilize the 2.5

M solution by passing it through a 0.22-µm filter. Store in 1-ml aliquots at 4°C.

deionized H2O, to 950 ml tryptone, 10 g yeast extract, 5 g NaCl, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1

2on liquid cycle.

liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm )

sterile H2O (cooled to 50°C or less), to 750 ml 5x M9 salts, 200 ml 1 M MgSO4, 2 ml

20% solution of the appropriate carbon source (e.g., 20% glucose), 20 ml 1 M CaCl2, 0.1 ml

sterile deionized H2O, to 980 ml

For solid medium, please see Media Containing Agar or Agarose

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Chapter 3 Working with bacteriophage M13 Vectors

If necessary, supplement the M9 minimal medium with stock solutions of the appropriate amino acids and vitamins.

(lac-proAB)] in the bacterial chromosome and the complementing proAB genes on the F' plasmid, supplement the M9 minimal medium with the following: 0.4% (w/v) glucose (dextrose) 5 mM MgSO4•7H2O 0.01% thiamine

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following: Bacto Agar (for plates) agarose (for plates)

15 g/liter 15 g/liter

Bacto Agar (for top agar) 7 g/liter agarose (for top agarose) 7 g/liter

and may boil over when swirled. Allow the medium to cool to 50-60°C before adding

thermolabile substances (e.g., antibiotics). To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored markers.

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Chapter 3 Working with bacteriophage M13 Vectors

bacteriophage plaques to spread and increases the chances of cross-contamination. This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37°C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.

MgSO4

To prepare a 1 M solution: Dissolve 12 g of MgSO4 in a final volume of 100 ml of H2O.

Sterilize by autoclaving or filter sterilization. Store at room temperature.

NaCl

to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl

solution at room temperature.

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume

Rich M13 LB YT

For solid medium, please see Media Containing Agar or Agarose.

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g NaCl, 5 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0

with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize

by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle.

Chapter 3, Protocol 3

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Chapter 3 Working with bacteriophage M13 Vectors

Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA

MATERIALS

Buffers and Solutions

Alkaline lysis solution II Alkaline lysis solution III Ethanol

Alkaline lysis solution I Phenol:chloroform (1:1, v/v)

TE (pH 8.0) containing 10 µg/ml RNase A

Enzymes and Buffers Restriction endonucleases

Vectors and Bacterial Strains

E. coli culture infected with bacteriophage M13

METHOD

1. Centrifuge 1 ml of the M13-infected cell culture at maximum speed for 5 minutes at

room temperature in a microfuge to separate the infected cells from the medium. Transfer the supernatant to a fresh microfuge tube and store at 4°C. Keep the infected bacterial cell pellet on ice.

The supernatant contains M13 bacteriophage housing single-stranded DNA. If desired, prepare M13 DNA from this supernatant at a later stage ( Chapter 3, Protocol 4 ). 2. Centrifuge the bacterial cell pellet for 5 seconds at 4°C and remove residual medium

with an automatic pipetting device.

3. Resuspend the cell pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous

vortexing.

Make sure that the bacterial pellet is completely dispersed in Alkaline lysis solution I. 4. Add 200 µl of freshly prepared Alkaline lysis solution II to the tube. Close the tube

tightly and mix by inverting the tube rapidly five times. Do not vortex. Store the tube on

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Chapter 3 Working with bacteriophage M13 Vectors

ice for 2 minutes after mixing.

5. Add 150 µl of ice-cold Alkaline lysis solution III to the tube. Close the tube to disperse

Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.

6. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge.

Transfer the supernatant to a fresh tube.

7. Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by

vortexing and then centrifuge the tube at maximum speed for 2-5 minutes. Transfer the aqueous (upper) phase to a fresh tube.

8. Precipitate the double-stranded DNA by adding 2 volumes of ethanol. Mix the contents

of the tube by vortexing and then allow the mixture to stand for 2 minutes at room temperature.

9. Recover the DNA by centrifugation at maximum speed for 5 minutes at 4°C in a

microfuge.

10. Remove the supernatant by gentle aspiration. Stand the tube in an inverted position on

a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube.

An additional ethanol precipitation step here helps to ensure that the double-stranded DNA is efficiently cleaved by restriction enzymes. • Dissolve the pellet of RF DNA in 100 µl of TE (pH 8.0).

• Add 50 µl of 7.5 M ammonium acetate, mix well, and add 300 µl of ice-cold ethanol. • Store the tube for 15 minutes at room temperature or overnight at -20°C and then collect the precipitated DNA by centrifugation at maximum speed for 5-10 minutes at 4°C in a microfuge. Remove the supernatant by gentle aspiration.

• Rinse the pellet with 250 µl of ice-cold 70% ethanol, centrifuge again for 2-3 minutes, and discard the supernatant.

• Allow the pellet of DNA to dry in the air for 10 minutes and then dissolve the DNA as described in Step 12.

11. Add 1 ml of 70% ethanol at 4°C and centrifuge again for 2 minutes. Remove the

supernatant as described in Step 10, and allow the pellet of nucleic acid to dry in the air for 10 minutes.

12. To remove RNA, resuspend the pellet in 25 µl of TE (pH 8.0) with RNase. Vortex

briefly.

13. Analyze the double-stranded RF DNA by digestion with appropriate restriction

endonucleases followed by electrophoresis through an agarose gel.

RECIPES

Alkaline Lysis Solution I

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Chapter 3 Working with bacteriophage M13 Vectors

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/cm) on liquid cycle, and store at 4°C.

For plasmid preparation.

Alkaline Lysis Solution II

0.2 N NaOH (freshly diluted from a 10 N stock) 1% (w/v) SDS

Prepare Solution II fresh and use at room temperature. For plasmid preparation.

Alkaline Lysis Solution III 5 M potassium acetate, 60.0 ml glacial acetic acid, 11.5 ml H2O, 28.5 ml

The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.

For plasmid preparation.

EDTA

To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

NaOH

breakage of glass containers. Prepare this solution with extreme care in plastic

The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As

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Chapter 3 Working with bacteriophage M13 Vectors

an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2O. Store the solution in a plastic container at room temperature. Sterilization is not necessary.

Potassium Acetate 5 M potassium acetate, 60 ml glacial acetic acid, 11.5 ml H2O, 28.5 ml

The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate.

Store the buffer at room temperature.

SDS

Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-grade SDS in 900 ml of H2O. Heat to 68°C and stir with a magnetic concentrated HCl. Adjust the volume to 1 liter with H2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.

stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of

100 mM Tris-Cl (desired pH) 10 mM EDTA (pH 8.0)

(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2

) on liquid cycle. Store the buffer at room temperature.

Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH HCl

70 ml 7.4 7.6 60 ml

8.0 42 ml

(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 liter with H2O. Dispense into aliquots and

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Chapter 3 Working with bacteriophage M13 Vectors

sterilize by autoclaving.

If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases approx. 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, and 8.6 at 5°C, 25°C, and 37°C, respectively.

CAUTIONS

Glacial acetic acid

inhalation, ingestion, or skin absorption. Wear appropriate gloves and goggles. Use in

a chemical fume hood.

Acetic acid (concentrated) must be handled with great care. It may be harmful by

NaOH

NaOH, see Sodium hydroxide

Phenol:chloroform

Phenol:chloroform, see Phenol; Chloroform

SDS

the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

REFERENCES

1. Birnboim H.C. and Doly J. 1979. A rapid extraction procedure for screening

recombinant plasmid DNA.Nucleic Acids Res. 7:1513-1523. 2. Ish-Horowicz D. and Burke J.F. 1981. Rapid and efficient cosmid cloning.Nucleic Acids

Res. 9:29-2998.

SDS (Sodium dodecyl sulfate) is toxic, an irritant, and poses a risk of severe damage to

Chapter 3, Protocol 4

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Chapter 3 Working with bacteriophage M13 Vectors

Preparation of Single-stranded Bacteriophage M13 DNA

MATERIALS

Buffers and Solutions Chloroform Ethanol Phenol

PEG 8000 (20% w/v) in 2.5 M NaCl Sodium acetate (3 M, pH 5.2) Gel-loading buffer IV TE (pH 8.0)

Nucleic Acids and Oligonucleotides

Single-stranded bacteriophage M13 vector of recombinant DNA

Vectors and Bacterial Strains

E. coli cultures infected with bacteriophage M13

Prepare an infected culture as described in Chapter 3, Protocol 2. These cultures should be infected with both the hoped-for recombinant bacteriophage and a control culture infected with nonrecombinant bacteriophage. E. coli cultures, uninfected

Prepare a mock-infected culture by picking an area of the E. coli lawn from the plate that does not contain a plaque as a negative control. Use this culture to monitor the recovery of bacteriophage M13 particles.

METHOD

1. Transfer 1 ml of the infected and uninfected cultures to separate microfuge tubes and

centrifuge the tubes at maximum speed for 5 minutes at room temperature. Transfer

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Chapter 3 Working with bacteriophage M13 Vectors

each supernatant to a fresh microfuge tube at room temperature.

2. To the supernatant add 200 µl of 20% PEG in 2.5 M NaCl. Mix the solution well by

inverting the tube several times, followed by gentle vortexing. Allow the tube to stand for 15 minutes at room temperature.

3. Recover the precipitated bacteriophage particles by centrifugation at maximum speed

for 5 minutes at 4°C in a microfuge.

4. Carefully remove all of the supernatant using a disposable pipette tip linked to a

vacuum line or a drawn-out Pasteur pipette attached to a rubber bulb. Centrifuge the tube again for 30 seconds and remove any residual supernatant.

A tiny, pinhead-sized, pellet of precipitated bacteriophage particles should be visible at the bottom of the tube. No pellet should be visible in the negative control tube in which a portion of the uninfected E. coli lawn was inoculated.

5. Resuspend the pellet of bacteriophage particles in 100 µl of TE (pH 8.0) by vortexing.

It is important to resuspend the bacteriophage pellet completely to allow efficient extraction of the single-stranded DNA by phenol in the next step.

6. To the resuspended pellet add 100 µl of equilibrated phenol. Mix well by vortexing for

30 seconds. Allow the sample to stand for 1 minute at room temperature, and then vortex for another 30 seconds.

7. Centrifuge the sample at maximum speed for 3-5 minutes at room temperature in a

microfuge. Transfer as much as is easily possible of the upper, aqueous phase to a fresh microfuge tube.

Do not try to transfer all of the aqueous phase. Much cleaner preparations of single-stranded DNA are obtained when approx. 5 µl of the aqueous phase is left at the interface.

8. Recover the M13 DNA by standard precipitation with ethanol in the presence of 0.3 M

sodium acetate. Vortex briefly to mix, and incubate the tubes for 15-30 minutes at room temperature or overnight at -20°C.

9. Recover the precipitated single-stranded bacteriophage DNA by centrifugation at

maximum speed for 10 minutes at 4°C in a microfuge.

10. Remove the supernatant by gentle aspiration, being careful not to disturb the DNA

pellet (which is often only visible as a haze on the side of the tube). Centrifuge the tube again for 15 seconds and remove any residual supernatant.

11. Add 200 µl of cold 70% ethanol and centrifuge at maximum speed for 5-10 minutes at

4°C. Immediately remove the supernatant by gentle aspiration.

At this stage, the pellet is not firmly attached to the wall of the tube. It is therefore important to work quickly and carefully to avoid losing the DNA.

12. Invert the open tube on the bench for 10 minutes to allow any residual ethanol to drain

and evaporate. Dissolve the pellet in 40 µl of TE (pH 8.0). Warm the solution to 37°C for 5 minutes to speed dissolution of the DNA. Store the DNA solutions at -20°C. The yield of single-stranded DNA is usually 5-10 µg/ml of the original infected culture. 13. Estimate the DNA concentration of a few of the samples by mixing 2-µl aliquots of the

DNA from Step 12 each with 1 µl of sucrose gel-loading buffer. Load the samples into the wells of a 1.2% agarose gel cast in 0.5x TBE and containing 0.5 µg/ml ethidium bromide. As controls, use varying amounts of M13 DNA preparations of known

Chapter 3 19

Chapter 3 Working with bacteriophage M13 Vectors

concentrations. Examine the gel after electrophoresis for 1 hour at 6 V/cm. Estimate the amount of DNA from the intensity of the fluorescence.

Usually 2-3 µl of a standard bacteriophage M13 DNA preparation is required for each set of four dideoxy cycle sequencing reactions using dye primers.

RECIPES

6x Gel-loading Buffer Alkaline Gel-loading Buffer 6x Gel-loading Buffer I 6x Gel-loading Buffer II 6x Gel-loading Buffer III 6x Gel-loading Buffer IV

6x Gel-loading Buffer I 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 40% (w/v) sucrose in H2O Store at 4°C.

6x Gel-loading Buffer II 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF

15% (w/v) Ficoll (Type 400; Pharmacia) in H2O Store at room temperature.

6x Gel-loading Buffer III 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 30% (v/v) glycerol in H2O Store at 4°C.

6x Gel-loading Buffer IV 0.25% (w/v) bromophenol blue

Chapter 3 20

Chapter 3 Working with bacteriophage M13 Vectors

40% (w/v) sucrose in H2O Store at 4°C.

Alkaline Gel-loading Buffer 300 mM NaOH 6 mM EDTA

18% (w/v) Ficoll (Type 400, Pharmacia) 0.15% (w/v) bromocresol green 0.25% (w/v) xylene cyanol For a 6x buffer.

EDTA

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

Ficoll 400 (20% w/v)

Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-µl aliquots at -20°C.

Gel-loading Buffer IV 6x Gel-loading Buffer

Glycerol

9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed

0.22-µm filter. Store in 200-ml aliquots at 4°C.

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in

NaOH

The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause

breakage of glass containers. Prepare this solution with extreme care in plastic

Chapter 3 21

Chapter 3 Working with bacteriophage M13 Vectors

beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2O. Store the solution in a plastic container at room temperature. Sterilization is not necessary.

PEG 8000

Working concentrations of PEG (polyethylene glycol) range from 13% to 40% (w/v). Prepare the appropriate concentration by dissolving PEG 8000 in sterile H2O, warming

if necessary. Sterilize the solution by passing it through a 0.22-µm filter. Store the solution at room temperature.

Phenol

Most batches of commercial liquified phenol are clear and colorless and can be used in molecular cloning without redistillation. Occasionally, batches of liquified phenol are pink or yellow, and these should be rejected and returned to the manufacturer. Crystalline phenol is not recommended because it must be redistilled at 160°C to remove oxidation products, such as quinones, that cause the breakdown of phosphodiester bonds or cause cross-linking of RNA and DNA.Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH. Wear gloves, full face protection, and a lab coat when carrying out this procedure.

1. Store liquified phenol at -20°C. As needed, remove the phenol from the freezer, allow it to warm to room temperature, and then melt it at 68°C. Add hydroxyquinoline to a final concentration of 0.1%. This compound is an antioxidant, a partial inhibitor of RNase, and a weak chelator of metal ions. In addition, its yellow color provides a 2. To the melted phenol, add an equal volume of buffer (usually 0.5 M Tris-Cl [pH 8.0] at room temperature). Stir the mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer, and when the two phases have separated, aspirate as much as possible of the upper (aqueous) phase using a glass pipette attached to a vacuum line equipped with appropriate traps.

3. Add an equal volume of 0. 1 M Tris-Cl (pH 8.0) to the phenol. Stir the mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer and remove the upper aqueous phase as described in Step 2. Repeat the extractions until the pH of the phenolic phase is >7.8 (as measured with pH paper).

4. After the phenol is equilibrated and the final aqueous phase has been removed, add 0.1 volume of 0.1 M Tris-Cl (pH 8.0) containing 0.2% at 4°C for periods of up to 1 month.

-mercaptoethanol. The phenol

solution may be stored in this form under 100 mM Tris-Cl (pH 8.0) in a light-tight bottle

convenient way to identify the organic phase.

Chapter 3 22

Chapter 3 Working with bacteriophage M13 Vectors

Sodium Acetate

To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate•3H2O in 800 ml of H2O. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.0 with dilute acetic

acid. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.

100 mM Tris-Cl (desired pH) 10 mM EDTA (pH 8.0)

(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2

) on liquid cycle. Store the buffer at room temperature.

Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH HCl 7.4 70 ml 7.6 60 ml 8.0 42 ml

(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.

CAUTIONS

Chloroform

tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid

breathing the vapors. Wear appropriate gloves and safety glasses. Always use in a Chloroform CHCl3 is irritating to the skin, eyes, mucous membranes, and respiratory

Chapter 3 23

Chapter 3 Working with bacteriophage M13 Vectors

chemical fume hood.

NaOH

NaOH, see Sodium hydroxide

PEG

PEG (Polyethylene glycol) may be harmful by inhalation, ingestion, or skin absorption.

Avoid inhalation of powder. Wear appropriate gloves and safety glasses.

Phenol

Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles, that come in contact with phenol with a large volume of water and wash with soap and water; do not use ethanol!

and protective clothing. Always use in a chemical fume hood. Rinse any areas of skin

REFERENCES

1. Messing J. 1983. New M13 vectors for cloning.Methods Enzymol. 101:20-78.

2. Sanger F., Coulson A.R., Barrell B.G., Smith A.J., and Roe B.A. 1980. Cloning in

single-stranded bacteriophage as an aid to rapid DNA sequencing.J. Mol. Biol. 143:161-178.

Chapter 3, Protocol 5

Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA

Chapter 3 Working with bacteriophage M13 Vectors

RECIPE

MATERIALS

Buffers and Solutions Ethanol NaCl (solid) Phenol

Phenol:chloroform (1:1, v/v) PEG 8000 (20% w/v) in H2O

Sodium acetate (3 M, pH 5.2) STE TE (pH 8.0)

Tris-Cl (10 mM, pH 8.0)

Media

Rich M13 medium containing 5 mM MgCl2

Transfer 250 ml of the medium into a 2-liter flask and warm to 37°C before Step 2.

Additional Reagents

Step 5 of this protocol requires the reagents listed in Chapter 1, Protocol 3 , Chapter 1, Protocol 8 , Chapter 1, Protocol 9 , and Chapter 1, Protocol 10 .

Vectors and Bacterial Strains E. coli F´ plating bacteria Bacteriophage M13 Stock

METHOD

1. Transfer 2.5 ml of plating bacteria (please see Chapter 3, Protocol 1 ) to a sterile tube

(13 x 100 mm or 17 x 100 mm). Add 0.5 ml of bacteriophage M13 stock (approx. 5 x 10 pfu) and mix by tapping the side of the tube. Incubate the infected cells for 5 minutes at room temperature.

2. Dilute the infected cells into 250 ml of fresh LB or YT medium containing 5 mM MgCl2

prewarmed to 37°C in a 2-liter flask. Incubate for 5 hours at 37°C with constant, vigorous shaking.

3. Harvest the infected cells by centrifugation at 4000g (5000 rpm in a Sorvall GSA rotor)

for 15 minutes at 4°C. Recover the supernatant, which may be used for large-scale preparations of single-stranded bacteriophage M13 DNA, as described in Steps 7-17

Chapter 3 25

Chapter 3 Working with bacteriophage M13 Vectors

below.

4. Resuspend the bacterial pellet in 100 ml of ice-cold STE. Recover the washed cells by

centrifugation at 4000g (5000 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. 5. Isolate the bacteriophage M13 closed circular RF DNA by the alkaline lysis method

(please see Chapter 1, Protocol 3 ). Scale up the volumes of lysis solutions appropriately. Purify the DNA either by precipitation with PEG, by column chromatography, or by equilibrium centrifugation in CsCl-ethidium bromide gradients. 6. Measure the concentration of the DNA spectrophotometrically and confirm its integrity

by agarose gel electrophoresis. Store the closed circular DNA in small (1-5 µg) aliquots at -20°C.

7. To isolate single-stranded DNA from the bacteriophage particles in the infected cell

medium, transfer the 250-ml supernatant from Step 3 to a 500-ml beaker containing a magnetic stirring bar.

8. Add 10 g of PEG and 7.5 g of NaCl to the supernatant. Stir the solution for 30-60

minutes at room temperature.

9. Collect the precipitate by centrifugation at 10,000g (7800 rpm in a Sorvall GSA rotor)

for 20 minutes at 4°C. Invert the centrifuge bottle for 2-3 minutes to allow the supernatant to drain, and then use Kimwipes to remove the last traces of supernatant from the walls and neck of the bottle.

Avoid touching the thin whitish film of precipitated bacteriophage particles on the side and bottom of the centrifuge bottle.

10. Add 10 ml of 10 mM Tris-Cl (pH 8.0) to the bottle. Swirl the solution in the bottle and

use a Pasteur pipette to rinse the sides of the bottle thoroughly. When the bacteriophage pellet is dissolved, transfer the solution to a 30-ml Corex centrifuge tube.

11. To the bacteriophage suspension, add an equal volume of equilibrated phenol, seal

the tube with a silicon rubber stopper, and mix the contents by vortexing vigorously for 2 minutes.

12. Centrifuge the solution at 3000g (5000 rpm in a Sorvall SS-34 rotor) for 5 minutes at

room temperature. Transfer the upper aqueous phase to a fresh tube and repeat the extraction with 10 ml of phenol:chloroform.

If there is a visible interface between the organic and aqueous layers, then extract the aqueous supernatant once more with chloroform.

13. Transfer equal amounts of the aqueous phase to each of two 30-ml Corex tubes. Add

0.5 ml of 3 M sodium acetate (pH 5.2) and 11 ml of ethanol to each tube. Mix the solutions well and then store them for 15 minutes at room temperature.

14. Recover the precipitate of single-stranded DNA by centrifugation at 12,000g (10,000

rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°C. Carefully remove all of the supernatant.

Most of the precipitated DNA will collect in a thin film along the walls of the centrifuge tubes.

15. Add 30 ml of 70% ethanol at 4°C to each tube, and centrifuge at 12,000g (10,000 rpm

in a Sorvall SS-34 rotor) for 10 minutes at 4°C. Carefully remove as much of the supernatant as possible, invert the tubes to allow the last traces of supernatant to

Chapter 3 26

Chapter 3 Working with bacteriophage M13 Vectors

drain away from the precipitate, and wipe the neck of the tubes with Kimwipes. 16. Allow the residual ethanol to evaporate at room temperature. Dissolve the pellets in 1

ml of TE (pH 8.0). Store the DNA at -20°C.

17. Measure the concentration of the DNA spectrophotometrically and confirm its integrity

by agarose gel electrophoresis. Store the closed circular DNA in small (10-50 µg) aliquots at -20°C.

RECIPES

EDTA

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

deionized H2O, to 950 ml tryptone, 10 g yeast extract, 5 g NaCl, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1

2on liquid cycle.

liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm )

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following:

Bacto Agar (for plates) 15 g/liter Bacto Agar (for top agar) 7 g/liter

agarose (for plates)

15 g/liter

agarose (for top agarose) 7 g/liter

Chapter 3 27

Chapter 3 Working with bacteriophage M13 Vectors

When the medium has hardened completely, invert the plates and store them at 4°C until needed. The plates should be removed from storage 1-2 hours before they are used. If the plates are fresh, they will \"sweat\" when incubated at 37°C. When this condensation drops on the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread and increases the chances of cross-contamination. This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37°C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.

NaCl

to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl

solution at room temperature.

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume

PEG 8000

Working concentrations of PEG (polyethylene glycol) range from 13% to 40% (w/v). Prepare the appropriate concentration by dissolving PEG 8000 in sterile H2O, warming

if necessary. Sterilize the solution by passing it through a 0.22-µm filter. Store the solution at room temperature.

Phenol

molecular cloning without redistillation. Occasionally, batches of liquified phenol are

Most batches of commercial liquified phenol are clear and colorless and can be used in pink or yellow, and these should be rejected and returned to the manufacturer.

Chapter 3 28

Chapter 3 Working with bacteriophage M13 Vectors

Crystalline phenol is not recommended because it must be redistilled at 160°C to remove oxidation products, such as quinones, that cause the breakdown of phosphodiester bonds or cause cross-linking of RNA and DNA.Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH. Wear gloves, full face protection, and a lab coat when carrying out this procedure.

2. To the melted phenol, add an equal volume of buffer (usually 0.5 M Tris-Cl [pH 8.0] at room temperature). Stir the mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer, and when the two phases have separated, aspirate as much as possible of the upper (aqueous) phase using a glass pipette attached to a vacuum line equipped with appropriate traps.

4. After the phenol is equilibrated and the final aqueous phase has been removed, add 0.1 volume of 0.1 M Tris-Cl (pH 8.0) containing 0.2% at 4°C for periods of up to 1 month.

-mercaptoethanol. The phenol

solution may be stored in this form under 100 mM Tris-Cl (pH 8.0) in a light-tight bottle

Rich M13 LB YT

For solid medium, please see Media Containing Agar or Agarose.

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/cm) on liquid cycle. Store the

sterile solution at 4°C.

Sodium Acetate

To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate•3H2O in 800 ml of H2O.

Chapter 3 29

Chapter 3 Working with bacteriophage M13 Vectors

Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.0 with dilute acetic acid. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.

100 mM Tris-Cl (desired pH) 10 mM EDTA (pH 8.0)

(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2

) on liquid cycle. Store the buffer at room temperature.

Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH HCl 7.4 70 ml 7.6 60 ml 8.0 42 ml

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g NaCl, 5 g

For solid medium, please see Media Containing Agar or Agarose.

with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize

by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle.

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0

Chapter 3 30

Chapter 3 Working with bacteriophage M13 Vectors

CAUTIONS

PEG

PEG (Polyethylene glycol) may be harmful by inhalation, ingestion, or skin absorption.

Avoid inhalation of powder. Wear appropriate gloves and safety glasses.

Phenol

Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles,

and protective clothing. Always use in a chemical fume hood. Rinse any areas of skin

that come in contact with phenol with a large volume of water and wash with soap and water; do not use ethanol!

Phenol:chloroform

Phenol:chloroform, see Phenol; Chloroform

Chapter 3, Protocol 6

Cloning into Bacteriophage M13 Vectors

MATERIALS

Buffers and Solutions ATP (10 mM) Ethanol

Chapter 3 31

Chapter 3 Working with bacteriophage M13 Vectors

IPTG (20% w/v)

Phenol:chloroform (1:1, v/v) Sodium acetate (3 M, pH 5.2) TE (pH 7.6 and pH 8.0) X-gal solution (2% w/v)

Enzymes and Buffers Bacteriophage T4 DNA ligase Restriction endonucleases

The choice of restriction enzymes to be used in Steps 1 and 6 depends on the cloning strategy.

Nucleic Acids and Oligonucleotides Foreign DNA

Individual fragments of foreign DNA to be cloned in M13 vectors are usually derived from a larger segment of DNA that has already been cloned and characterized in another vector. Test DNA

Media

Rich M13 agar plates Rich M13 medium

Rich M13 top agar or agarose

Additional Reagents

Step 4 of this protocol requires the reagents listed in Chapter 1, Protocol 20 .

Vectors and Bacterial Strains Bacteriophage M13 vector DNA (RF)

E. coli competent cells of an appropriate strain carrying an F´ plasmid

Competent cells may be prepared in the laboratory as described in Chapter 1, Protocol 25 or purchased from commercial suppliers. E. coli F´ plating bacteria

Plating bacteria may be prepared in the laboratory as described in Chapter 3, Protocol 1 or purchased from commercial suppliers.

METHOD

1. Digest 1-2 µg of the bacteriophage M13 vector RF DNA to completion with a three- to

fivefold excess of the appropriate restriction enzyme(s). Set up a control reaction containing M13 RF DNA but no restriction enzyme(s).

Chapter 3 32

Chapter 3 Working with bacteriophage M13 Vectors

2. At the end of the incubation period, remove a small sample of DNA (50 ng) from each

of the reactions and analyze the extent of digestion by electrophoresis through an 0.8% agarose gel. If digestion is incomplete (i.e., if any closed circular DNA is visible), add more restriction enzyme(s) and continue the incubation.

3. When digestion is complete, purify the M13 DNA by extraction with phenol:chloroform

followed by standard precipitation with ethanol in the presence of 0.3 M sodium acetate (pH 5.2). Dissolve the DNA in TE (pH 8.0) at a concentration of 50 µg/ml. 4. If required, dephosphorylate the linearized vector DNA by treatment with calf alkaline

phosphatase or shrimp alkaline phosphatase. At the end of the dephosphorylation reaction, inactivate the alkaline phosphatase by heat and/or by digestion with proteinase K, followed by extraction with phenol:chloroform (for details, please see Chapter 1, Protocol 20 ).

5. Recover the linearized M13 DNA as outlined in Step 3. Dissolve the dephosphorylated

DNA in TE (pH 7.6) at a concentration of 50 µg/ml.

6. Generate individual restriction fragments of foreign DNA by cleavage with the

appropriate restriction enzymes and purify them by agarose gel electrophoresis. Dissolve the final preparation of foreign DNA in TE (pH 7.6) at a concentration of 50 µg/ml.

When ligating DNAs with complementary cohesive termini, please follow Steps 7-9 below. For methods to set up blunt-ended ligation reactions, please see Chapter 1, Protocol 19.

7. In a microfuge tube (Tube A), mix together approx. 50 ng of vector DNA and a one- to

fivefold molar excess of the target (foreign) DNA fragment(s). The combined volume of the two DNAs should not exceed 8 µl. If necessary, add TE (pH 7.6) to adjust the volume to 7.5-8.0 µl. As controls, set up three ligation reactions containing: Tube DNA B C D

the same amount of vector DNA, but no foreign DNA

the same amount of vector DNA and a one- to fivefold molar excess of the target DNA fragment(s) the same amount of vector DNA together with an equal amount by weight of a test DNA that has been successfully cloned into bacteriophage M13 on previous occasions

As a test DNA, we routinely use a standard preparation of bacteriophage generate termini that are complementary to the M13 vectors to be used.

9. Add 1 µl of 10x ligation buffer and 1 µl of 10 mM ATP to all four reactions (Tubes A-D).

Omit ATP if using a commercial buffer that contains ATP.

10. Add 0.5 Weiss unit of bacteriophage T4 DNA ligase to Tubes A, B, and D. Mix the

components by gently tapping the side of each tube for several seconds. Incubate the

Chapter 3 33

DNA

cleaved with restriction enzymes that recognize tetranucleotide sequences and

Chapter 3 Working with bacteriophage M13 Vectors

ligation reactions for 4-16 hours at 12-16°C.

At the end of the ligation reaction, analyze 1 µl of each ligation reaction by electrophoresis through an 0.8% agarose gel. Bands of circular recombinant molecules containing vector and fragment(s) of foreign DNA should be visible in the test reaction (Tube A) but not in the control (Tube C).

After ligation, the reactions may be stored at -20°C until transformation.

11. Prepare and grow an overnight culture of plating bacteria (please see Chapter 3, Protocol 1 ) in YT or LB medium at 37°C with constant shaking.

12. Remove from the -70°C freezer an aliquot of frozen competent cells of the desired

strain carrying an F´ plasmid, allow the cells to thaw at room temperature, and then place them on ice for 10 minutes.

13. Transfer 50-100 µl of the competent F´ bacteria to each of 16 sterile 5-ml culture tubes

(Falcon 2054, Becton Dickinson) that have been chilled to 0°C.

14. Immediately add 0.1-, 1.0-, and 5-µl aliquots of the ligation reactions and controls

(Tubes A-D) to separate tubes of competent cells. Mix the DNAs with the bacteria by tapping the sides of the tubes gently for a few seconds. Store on ice for 30-40 minutes. Include two transformation controls, one containing 5 pg of bacteriophage M13 RF DNA and the other containing no added DNA.

15. While the ligated DNA is incubating with the competent cells, prepare a set of 16

sterile culture tubes containing 3 ml of melted YT or LB top agar. Store the tubes at 47°C in a heating block or water bath until needed in Step 16.

16. Transfer the tubes containing the competent bacteria and DNA to a water bath

equilibrated to 42°C. Incubate the tubes for exactly 90 seconds. Immediately return the tubes to an ice-water bath.

17. Add 40 µl of 2% X-gal, 4 µl of 20% IPTG, and 200 µl of the overnight culture of E. coli

cells (Step 10) to the tubes containing the melted top agar prepared in Step 14, and mix the contents of the tubes by gentle vortexing for a few seconds. Transfer each sample of the transformed bacteria to the tubes. Cap the tubes and mix the contents by gently inverting the tubes three times. Pour the contents of each tube in turn onto a separate labeled LB agar plate. Swirl the plate to ensure an even distribution of bacteria and top agar.

18. Close the plates and allow the top agar to harden for 5 minutes at room temperature.

Use a Kimwipe to remove any condensation from the top of the plate, invert the plates, and incubate at 37°C.

Plaques will be fully developed after 8-12 hours.

RECIPES

ATP

Dissolve an appropriate amount of solid ATP in 25 mM Tris-Cl (pH 8.0).

Chapter 3 34

Chapter 3 Working with bacteriophage M13 Vectors

Store the 10 mM ATP solution in small aliquots at -20°C.

EDTA

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

IPTG

IPTG is isopropylthio--D-galactoside. Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled H2O. Adjust the volume of the solution to 10 ml

with H2O and sterilize by passing it through a 0.22-µm disposable filter. Dispense the solution into 1-ml aliquots and store them at -20°C.

deionized H2O, to 950 ml tryptone, 10 g yeast extract, 5 g NaCl, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1

2on liquid cycle.

liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm )

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following: Bacto Agar (for plates) agarose (for plates)

15 g/liter 15 g/liter

Bacto Agar (for top agar) 7 g/liter

agarose (for top agarose) 7 g/liter

Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle. When

Chapter 3 35

Chapter 3 Working with bacteriophage M13 Vectors

the medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Allow the medium to cool to 50-60°C before adding thermolabile substances (e.g., antibiotics). To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored markers.

NaCl

to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl

solution at room temperature.

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume

Rich M13 LB YT

For solid medium, please see Media Containing Agar or Agarose.

Sodium Acetate

To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate•3H2O in 800 ml of H2O. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.0 with dilute acetic

acid. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.

Chapter 3 36

Chapter 3 Working with bacteriophage M13 Vectors

100 mM Tris-Cl (desired pH) 10 mM EDTA (pH 8.0)

(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2

) on liquid cycle. Store the buffer at room temperature.

Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH HCl 7.4 70 ml 7.6 60 ml 8.0 42 ml

X-gal Solution

X-gal is 5-bromo-4-chloro-3-indolyl--D-galactoside. Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use

a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to

prevent damage by light and store at -20°C. It is not necessary to sterilize X-gal solutions by filtration.

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g NaCl, 5 g

For solid medium, please see Media Containing Agar or Agarose.

Chapter 3 37

Chapter 3 Working with bacteriophage M13 Vectors

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0 by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle.

with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize

CAUTIONS

Phenol:chloroform

Phenol:chloroform, see Phenol; Chloroform

Radioactive substances

Radioactive substances: When planning an experiment that involves the use of radioactivity, consider the physico-chemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and 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 short wavelengths either generated by technical devices or emitted by radioactive materials. They might be emitted isotropically from

the source or may be focused into a beam. Their potential dangers depend on the time period of exposure, the intensity experienced, and the wavelengths used. Be aware that appropriate shielding 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 office for further guidance in the appropriate use and disposal of radioactive materials. Always monitor thoroughly after using radioisotopes. A convenient calculator to perform routine radioactivity calculations can be found at: http://www.graphpad.com/calculators/radcalc.cfm.

X-gal

powder. Note that stock solutions of X-gal are prepared in DMF, an organic solvent. For

details, see DMF. See also BCIG.

X-gal may be toxic to the eyes and skin. Observe general cautions when handling the

Chapter 3 38

Chapter 3 Working with bacteriophage M13 Vectors

Chapter 3, Protocol 7

Analysis of Recombinant Bacteriophage M13 Clones

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. CAUTION RECIPE

MATERIALS

Buffers and Solutions SDS (2% w/v) 20x SSC

Gel-loading buffer IV

Nucleic Acids and Oligonucleotides

Single-stranded recombinant bacteriophage M13 DNA

Choose previously characterized recombinants that carry foreign sequences of known size to use as positive controls during gel electrophoresis.

Additional Reagents

Step 1 of this protocol requires the reagents listed in Chapter 3, Protocol 2.

Step 7 of this protocol may require the reagents listed in Chapter 2, Protocol 21 and Chapter 2, Protocol 22.

Vectors and Bacterial Strains

Bacteriophage M13 recombinant plaques in top agarose Prepared as described in Chapter 3, Protocol 6 .

Bacteriophage M13 nonrecombinant vector, grown as well-isolated plaques in top agarose E. coli F´ strain

METHOD

1. Prepare stocks of putative recombinant bacteriophages from single plaques, grown in

an appropriate F´ host, as described in Chapter 3, Protocol 2 .

As controls, prepare stocks of several nonrecombinant bacteriophages (picked from well-isolated dark blue plaques).

2. Use a micropipettor with a sterile tip to transfer 20 µl of each of the supernatants into a

fresh microfuge tube. Store the remainder of the supernatants at 4°C until needed.

Chapter 3 39

Chapter 3 Working with bacteriophage M13 Vectors

3. To each 20-µl aliquot of supernatant, add 1 µl of 2% SDS. Tap the sides of the tubes to

mix the contents, and then incubate the tubes for 5 minutes at 65°C.

4. To each tube, add 5 µl of sucrose gel-loading buffer. Again mix the contents of the

tubes by tapping and then analyze each sample by electrophoresis through an 0.7% agarose gel. Run the gel at 5 V/cm. As positive controls, use single-stranded DNA preparations of previously characterized M13 recombinants that carry foreign sequences of known size.

5. When the bromophenol blue has traveled the full length of the gel, photograph the

DNA under UV illumination.

6. Compare the electrophoretic mobilities of the single-stranded DNAs liberated from the

putative recombinants with those of the DNAs liberated from the control nonrecombinant bacteriophages.

The single-stranded DNAs of recombinants carrying sequences of foreign DNA longer than 200-300 nucleotides migrate slightly more slowly than empty vector through 0.7% agarose gels. Once recombinants of the desired size have been identified, single-stranded DNAs can be prepared from supernatants stored at 4°C (Step 2). 7. If necessary, confirm the presence of foreign DNA sequences by transferring

single-stranded DNAs from the gel to a nitrocellulose or nylon membrane (please see Chapter 2, Protocol 21 ) and hybridizing to an appropriate radiolabeled probe (please see Chapter 2, Protocol 22 ). Soak the gel in 10 volumes of 20x SSC for 45 minutes, and then transfer the DNA directly to the membrane.

RECIPES

6x Gel-loading Buffer Alkaline Gel-loading Buffer 6x Gel-loading Buffer I 6x Gel-loading Buffer II 6x Gel-loading Buffer III 6x Gel-loading Buffer IV

6x Gel-loading Buffer I 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 40% (w/v) sucrose in H2O Store at 4°C.

6x Gel-loading Buffer II 0.25% (w/v) bromophenol blue

Chapter 3 40

Chapter 3 Working with bacteriophage M13 Vectors

0.25% (w/v) xylene cyanol FF

15% (w/v) Ficoll (Type 400; Pharmacia) in H2O Store at room temperature.

6x Gel-loading Buffer III 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 30% (v/v) glycerol in H2O Store at 4°C.

6x Gel-loading Buffer IV 0.25% (w/v) bromophenol blue 40% (w/v) sucrose in H2O Store at 4°C.

Alkaline Gel-loading Buffer 300 mM NaOH 6 mM EDTA

18% (w/v) Ficoll (Type 400, Pharmacia) 0.15% (w/v) bromocresol green 0.25% (w/v) xylene cyanol For a 6x buffer.

EDTA

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

Ficoll 400 (20% w/v)

Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-µl aliquots at

Chapter 3 41

Chapter 3 Working with bacteriophage M13 Vectors

-20°C.

Gel-loading Buffer IV 6x Gel-loading Buffer

Glycerol

9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed

0.22-µm filter. Store in 200-ml aliquots at 4°C.

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in

NaOH

The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause breakage of glass containers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As

SDS

stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of

SSC

For 20x solution: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2O. Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume

to 1 liter with H2O. Dispense into aliquots. Sterilize by autoclaving. The final concentrations of the ingredients are 3.0 M NaCl and 0.3 M sodium acetate.

CAUTIONS

NaOH

NaOH, see Sodium hydroxide

Chapter 3 42

Chapter 3 Working with bacteriophage M13 Vectors

SDS

the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

SDS (Sodium dodecyl sulfate) is toxic, an irritant, and poses a risk of severe damage to

Chapter 3, Protocol 8

Producing Single-stranded DNA with Phagemid Vectors

MATERIALS

Buffers and Solutions Kanamycin (10 mg/ml) SDS (2% w/v)

Gel-loading buffer IV

Media

M9 minimal agar plates, supplemented biosynthetic operon (

This medium is needed when using E. coli strains that carry a deletion of the proline

[lac-proAB]) in the bacterial chromosome and the complementing

proAB genes on the F´ plasmid.

YT agar plates containing 60 µg/ml ampicillin 2x YT

2x YT containing 60 µg/ml ampicillin 2x YT containing 60 µg/ml kanamycin

Additional Reagents

Steps 2 and 5 of this protocol require the reagents listed in Chapter 3, Protocol 1 . Step 14 of this protocol requires the reagents listed in Chapter 3, Protocol 4 .

Chapter 3 43

Chapter 3 Working with bacteriophage M13 Vectors

Vectors and Bacterial Strains Bacteriophage M13K07 (helper)

M13K07 may be obtained commercially (e.g., from Pharmacia or New England Biolabs) and propagated as described in Steps 1-3 below. Store stocks of helper virus at 4°C in growth medium or at -20°C in growth medium containing 50% (v/v) glycerol. E. coli F´ strain E. coli strain DH11S

DH11S should be plated on supplemented minimal agar plates.

E. coli strain DH11S, transformed with bacteriophage M13 phagemid vector

Transform E. coli with the phagemid vector as described in Chapter 3, Protocol 6 . The transformed strain may be propagated as a culture as described in Chapter 3, Protocol 2 . E. coli strain DH11S, transformed with bacteriophage M13 recombinant phagemid vector clone carrying foreign DNA

Transform E. coli with the recombinant phagemid vector as described in Chapter 3, Protocol 6 . The transformed strain may be propagated as a culture as described in Chapter 3, Protocol 2 .

METHOD

1. In 20 ml of 2x YT medium, establish a culture of E. coli strain DH11S from a single

colony freshly picked from supplemented minimal agar plates. Incubate the culture at 37°C with moderate agitation until the OD600 reaches 0.8.

2. Prepare a series of tenfold dilutions of bacteriophage M13K07 in 2x YT medium, and

plate aliquots of the bacteriophage as described in Chapter 3, Protocol 1 to obtain well-isolated plaques on a lawn of DH11S cells.

3. Pick well-separated, single plaques and place each plaque in 2-3 ml of 2x YT medium

containing kanamycin (25 µg/ml) in a 15-ml culture tube. Incubate the infected cultures for 12-16 hours at 37°C with moderate agitation (250 cycles/minute).

Kanamycin is used in this protocol to ensure that all bacterial cells containing a phagemid genome are infected by the helper M13K07 bacteriophage. During propagation of M13K07 (e.g., Steps 1-3), there is selection for bacteriophage genomes that have lost the p15A origin and the Tn903 transposon. For this reason, it is essential to include kanamycin in the medium used to prepare the stock of helper virus in this step.

IMPORTANT Use stocks of M13K07 derived from single freshly picked plaques in the following steps.

4. Transfer the infected cultures to 1.5-ml sterile microfuge tubes and centrifuge them at

maximum speed for 2 minutes at 4°C in a microfuge. Transfer the supernatants to fresh tubes and store them at 4°C.

5. Measure the titer of each of the bacteriophage stocks by plaque formation ( Chapter 3, Protocol 1 ) on a strain of E. coli F´ (TG1, DH11S, NM522, or XL1-Blue) that supports

Chapter 3 44

Chapter 3 Working with bacteriophage M13 Vectors

the growth of bacteriophage M13.

The titer of infectious bacteriophage particles in the stocks should be 10 pfu/ml. Discard any stock with a lower titer.

6. Streak DH11S cells transformed by (i) the recombinant phagemid and (ii) the empty

(parent) phagemid vector onto two separate YT agar plates containing 60 µg/ml ampicillin. Incubate the plates for 16 hours at 37°C.

7. Pick (i) several colonies transformed by the recombinant phagemid and (ii) one or two

colonies transformed by the parent vector into sterile 15-ml culture tubes that contain 2-3 ml of 2x YT medium containing 60 µg/ml ampicillin.

8. To each culture, add M13K07 helper bacteriophage to achieve a final concentration of

2 x 10 pfu/ml. Incubate the cultures for 1.0-1.5 hours at 37°C with strong agitation (300 cycles/ minute).

9. Add kanamycin to the cultures to a final concentration of 25 µg/ml. Continue

incubation for a further 14-18 hours at 37°C.

10. Transfer the cell suspensions to microfuge tubes and separate the bacterial cells from

the growth medium by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge. Transfer the supernatants to fresh tubes and store them at 4°C.

11. Combine 40 µl of each supernatant with 2 µl of 2% SDS in 0.5-ml microfuge tubes. Mix

the contents of the tubes by tapping and then incubate the tubes for 5 minutes at 65°C.

12. Add 5 µl of sucrose gel-loading buffer to each sample of the phagemid DNA, mix the

samples, and load them into separate wells of an 0.7% agarose gel.

13. Carry out electrophoresis for several hours at 6 V/cm until the bromophenol blue has

migrated approximately half the length of the gel. Examine and photograph the gel by UV light.

Yields vary depending on the size and nature of foreign DNA in the phagemid, but are generally approx. 1 µg/ml of culture volume.

14. Isolate single-stranded phagemid DNA from the supernatants containing the largest

amount of single-stranded DNA. Follow the steps outlined in Chapter 3, Protocol 4 , scaling up the volumes two- to threefold.

RECIPES

5x M9 Salts Na2HPO4•7H2O, g KH2PO4, 15 g NaCl, 2.5 g NH4Cl, 5.0 g

deionized H2O, to 1 liter

Chapter 3 45

Chapter 3 Working with bacteriophage M13 Vectors

Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes

at 15 psi (1.05 kg/cm 2

) on liquid cycle.

6x Gel-loading Buffer Alkaline Gel-loading Buffer 6x Gel-loading Buffer I 6x Gel-loading Buffer II 6x Gel-loading Buffer III 6x Gel-loading Buffer IV

6x Gel-loading Buffer I 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 40% (w/v) sucrose in H2O Store at 4°C.

6x Gel-loading Buffer II 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF

15% (w/v) Ficoll (Type 400; Pharmacia) in H2O Store at room temperature.

6x Gel-loading Buffer III 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 30% (v/v) glycerol in H2O Store at 4°C.

6x Gel-loading Buffer IV 0.25% (w/v) bromophenol blue 40% (w/v) sucrose in H2O Store at 4°C.

Alkaline Gel-loading Buffer

Chapter 3 46

Chapter 3 Working with bacteriophage M13 Vectors

300 mM NaOH 6 mM EDTA

18% (w/v) Ficoll (Type 400, Pharmacia) 0.15% (w/v) bromocresol green 0.25% (w/v) xylene cyanol For a 6x buffer.

CaCl2

Dissolve 11 g of CaCl2•6H2O in a final volume of 20 ml of distilled H2O. Sterilize the 2.5

M solution by passing it through a 0.22-µm filter. Store in 1-ml aliquots at 4°C.

EDTA

of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt

Ficoll 400 (20% w/v)

Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-µl aliquots at -20°C.

Gel-loading Buffer IV 6x Gel-loading Buffer

Glycerol

9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed

0.22-µm filter. Store in 200-ml aliquots at 4°C.

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in

sterile H2O (cooled to 50°C or less), to 750 ml 5x M9 salts, 200 ml 1 M MgSO4, 2 ml

Chapter 3 47

Chapter 3 Working with bacteriophage M13 Vectors

20% solution of the appropriate carbon source (e.g., 20% glucose), 20 ml 1 M CaCl2, 0.1 ml

sterile deionized H2O, to 980 ml

For solid medium, please see Media Containing Agar or Agarose

If necessary, supplement the M9 minimal medium with stock solutions of the appropriate amino acids and vitamins.

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following: Bacto Agar (for plates) agarose (for plates)

15 g/liter 15 g/liter

Bacto Agar (for top agar) 7 g/liter agarose (for top agarose) 7 g/liter

2 Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle. When

Chapter 3 48

Chapter 3 Working with bacteriophage M13 Vectors

markers.

MgSO4

To prepare a 1 M solution: Dissolve 12 g of MgSO4 in a final volume of 100 ml of H2O.

Sterilize by autoclaving or filter sterilization. Store at room temperature.

NaCl

to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl

solution at room temperature.

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume

NaOH

SDS

stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of

Chapter 3 49

Chapter 3 Working with bacteriophage M13 Vectors

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g NaCl, 5 g

For solid medium, please see Media Containing Agar or Agarose.

with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize

by autoclaving for 20 minutes at 15 psi (1.05 kg/cm ) on liquid cycle.

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0

CAUTIONS

NaOH

NaOH, see Sodium hydroxide

SDS

the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

SDS (Sodium dodecyl sulfate) is toxic, an irritant, and poses a risk of severe damage to

Chapter 3 50

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