[转移贴]Preparation of Single-stranded Bacteriophage M13 DNA

hantavirus

原贴由jbclinnic发表于 2009-5-21 16:55

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 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-strandedDNA, 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 is 5-10 μg/ml infected culture.


MATERIALS

E. coli cultures infected with bacteriophage M13


Prepare an infected culture as described in Growing Bacteriophage M13 in Liquid Culture. These cultures should be infected with both the hoped-for recombinantbacteriophage 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.


Chloroform


Optional, please see Step 7.


Ethanol


Optional, please see Step 5.


Gel-loading buffer IV

PEG 8000 (20% w/v) in 2.5 M NaCl

Phenol

Single-stranded bacteriophage M13 vector of recombinant DNA

Sodium acetate (3 M, pH 5.2)

TE (pH 8.0)


METHOD


1. Transfer 1 ml of the infected and uninfected cultures to separate microcentrifuge tubes and centrifuge the tubes at maximum speed for 5 minutes at room temperature. Transfer each supernatant to a fresh microcentrifuge 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 microcentrifuge.


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


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 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.



REFERENCES

1. 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.[Medline]

2. Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol. 101: 20–78.[Medline]