Uracil-containing single-stranded phage M13 DNA preparation experiments
The classical Kunfcel oligonucleotide-directed mutagenesis method utilizes the selective action of uracil DNA glycosylase in E. coli to specifically screen out uracil base-containing DNA (see the Oligonucleotide Mutagenesis Information column). This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Uracil-containing single-stranded phage M13 DNA preparation experiments
Materials and Instruments
Buffers and Solutions Ethanol Sodium Chloride Phenol Sodium Acetate TE Agarose Gel Original Recombinant M13 Phage Single Stranded DNA YT Medium Move makings For more product details, please visit Aladdin Scientific website.
Corex Centrifuge Tubing Pasteur Dropper Column Chromatography Resin E. coli Strain CJ236 E. coli Strain TG1 JM109 or Equivalent
Buffers and solutions
Refer to Appendix 1 for various storage solutions, buffers and reagent compositions.
Dilute the stock solution to the desired concentration.
Ethanol
Sodium chloride (2.5 mol/L) Contains 15% Polyethylene glycol (m/V, PEG8000)
Phenol (pH 8.0)
Phenol:Chloroform (1:1,V/V)
Sodium acetate(3mol/L,pH5.2)
TE(pH7.6)
Gel
Agarose gel
See step 16.
Nucleic Acids and Oligonucleotides
DNA Molecular Quality Standard, original recombinant M13 phage single-stranded DNA
Medium
2xYT Medium
2XYT medium with 0.25ug/ml uracil
Centrifuge and rotor
SorvallGSA rotor or equivalent
SorvallSS-34 rotor or equivalent
Specialized equipment
Corex centrifuge tubes (15 ml and 30 ml)
Pasteur Dropper
Column Chromatography Resins
Use pre-packaged resins such as SephacrylS-400 (Pharmacia) or micro-preparation centrifuge columns (Promega).
60°C water bath
Other reagents
The reagents required for Step 1 of this program are listed in Chapter 3, Programs 3, 4, and 6.
The reagents required in Step 6 of this protocol are listed in Chapter 3, Scheme 1.
Vectors and Strains
See Appendix 3.
E. coli strain CJ236 (dut-ung-F')
See Kunkel (1985) and Kunkel (1987) for detailed information.
E. coli strains TG1, JM109 or equivalents
See step 6.
Methods
1. Mutagenesis preparation
a. Clone a small fragment of DNA (<500bp) carrying the target sequence into an appropriate M13 phage vector, e.g. M13 mp l8 or mp l9. b. Clone a small fragment of DNA (<500bp) carrying the target sequence into an appropriate M13 phage vector.
b. Isolate single-stranded template DNA and double-stranded replicative DNA from a newly grown phage spot produced by a recombinant phage.
Methods of cloning using M13 phage vectors and preparation of single-stranded and replicative phage DNA are described in Scenarios 3, 4 and 6 in Chapter 3.
c. Restriction endonuclease analysis of replicative DNA and single-stranded DNA sequence analysis methods to identify the correct recombinant clone.
The Kunkel process can also be used to generate uracil-incorporated single-stranded DNA from phage vectors (McClary et al. 1989, Wang et al. 1989, Liuetal. 1990, reviewed in Hagemeier l996). In this paper, E. coli CJ236 (or other strains with the phenotype dut-ung-F') was first transformed to ampicillin resistance with double-stranded phage DNA containing the target sequence. Repeated infection of bacteria containing target sequence phage DNA by helper phages converts the replicated form of phage DNA from double-stranded DNA to single-stranded DNAs (see Scheme 8 in Chapter 3). As it grows in uridine medium, some of the single-stranded DNA doped with uracil at the thymine position is packaged into viral particles and secreted into the culture medium. Isolation of this DNA (as described in step 5) is used as a template for targeted mutagenesis (Scheme 2). The use of phage vectors does not require that the starting target DNA be cloned into the M13 phage vector prior to targeted mutagenesis.
2. Transfer a single phage spot produced by the recombinant M13 phage into a microcentrifuge tube containing 1 ml of 2XYT culture medium using a sterilized Pasteur dropper.
3. Incubate at 60°C for 5 min to inactivate the bacterial cells (it is important to kill the bacterial cells to prevent continued production of thymidine-containing viral DNA during the next round of phage multiplication). Shake the tube vigorously for 30 s to release the phage encapsulated in the top layer of agar. ultracentrifuge at 4°C for 2 min to remove dead bacteriophage and cellular debris.
4. Transfer 50ul of supernatant to a 500 ml conical flask containing 50 ml 2xYT culture medium (supplemented with 0.25ug/ml uracil). It is not necessary to replenish the culture medium with thymine and adenosine as originally described by Kunkel (1985). Add 5 ml of mid-logarithmic E. coli CJ236 ( dut- ung- F') culture. Incubate at 37°C with vigorous shaking (300r/min) for 6 h. Efficient aeration is essential for phage growth.
Phage suspensions for inoculation were typically 109 and 1010 pfu/ml. mid-logarithmic E. coli cultures contained approximately 5X108 bacteria/ml. the low reproducibility of virulence (0.02-0.2 pfu/ml) ensured that the vast majority of phages were produced from dut- ung- F' strains of E. coli.
5. Cells were collected by centrifugation at 5000 g (6470r/min in Sorvall SS-34 rotor) for 30 min at 4°C. Transfer the supernatant to a new 250 ml centrifuge vial suitable for Sorvall GSA rotor or equivalent.
6. Determine the relative titer of the phage suspension on E. coli strains CJ236 ( dut- ung- F'), JM109, or TG1 (see Protocol 1 in Chapter 3). The titer of the phage in strain CJ236 should be 4 to 5 orders of magnitude higher than in the dut+ung+ strain.
To save time, most researchers start purifying phage particles before obtaining titer results. However, if purification is delayed, the crude phage suspension should be kept on ice.
Phage yield varies with recombinant. Typical titers of virus particles on a dut- ung- F' strain of E. coli (CJ236) are 5X1010 to 1x1011pfu/ml. however, poorly grown recombinants may achieve titers of only 1X1010 to 2X1010pfu/ml. if the yield of single-stranded DNA is insufficient, this can be remedied by either of two ways.
-Determine the titer of the phage reservoir solution used for infection in the dut-ung-F' E. coli strain. Adjust the volume of the inoculum to 0.1 pfu/fine seedling cell to make it conducive to repeat infections. The infected cultures were incubated for 6 h.
Infected cultures are grown for 12 h instead of 6 h. As described in Chapter 5, various deletions may grow faster than wild-type recombinants during extended incubation, so it is desirable to confirm that most of the molecular size of the single-stranded DNA used as a template is correct. The method for analyzing M13 phage DNA by gel electrophoresis can be found in Scheme 1 in Chapter 3.
7. Measure the volume of the phage suspension and add 0.25 times the volume of 2.5 mol/L NaCl containing 15% (m/V) PAGE 8000, shake the centrifuge bottle to mix the reaction, and place the bottle on ice for 1 h. The reaction will be analyzed by gel electrophoresis.
Recover the precipitated phage pellet by centrifugation at 8.4°C for 20 min at 5000 g (5500 r/min, Sorvall GSA rotor). The supernatant was aspirated by vacuum, and the centrifuge bottle was inverted to allow the residual supernatant to drain completely. A burette connected to a vacuum extraction device was used to remove all liquid trapped on the wall of the centrifuge tube.
9. Suspend the phage precipitate in 4 ml of TE buffer (pH 7.6). Transfer the suspension to a 15 ml Corex centrifuge tube. Wash the walls of the centrifuge tube with an additional 2 ml of TE Buffer (pH 7.6) and combine the washings into the Corex tube. Vigorously shake the suspension for 30s and place the tube on ice for 1h.
10. Vigorously shake the phage suspension for 30 s, then centrifuge at 5000 g (6470 r/min, Sorvall SS-34 rotor) for 20 min at 4°C to collect the bacterial debris at the bottom of the tube.
11. Carefully transfer the supernatant obtained into a 15 ml polypropylene centrifuge tube, transferring the supernatant so as not to pick up the bacterial debris precipitate. Extract the suspension twice with phenol (pH 8.0) and once with phenol:chloroform. Separate the phases by centrifugation at 4000 g (5800 r/min with Sorvall SS-34 rotor) for 5 min at room temperature. Avoid transferring interfacial substances.
12. Transfer the aqueous phase from the last extraction to a glass centrifuge tube (e.g. 30 ml Corex tube). Measure the volume of the solution and add 0.1 times the volume of NaCl (pH 5.2), and 2 times the volume of 0°C pre-cooled anhydrous ethanol. Mix the contents thoroughly and place the tube on ice for 30 min.
Recover the DNA by centrifugation at 5000 g (6470r/min, Sorvall SS-34 rotor) for 20 min at 13.4°C. Carefully remove the supernatant from the centrifuge. Add 10 ml of 70% ethanol kept at room temperature. Vortex slightly and centrifuge again.
14. Carefully aspirate the supernatant and invert the tube at room temperature until the ethanol has completely evaporated. Dissolve the DNA in 200ul of TE (pH 7.6) buffer.
15. Purify the uracil-containing, single-stranded M13 phage DNA from the suspension using a centrifugal chromatography column capable of excluding large molecules of DNA (>100 nucleotides) as described in Appendix 8.
Single-stranded M13 phage DNA propagated in a dut- ung- F' E. coli strain was purified by centrifugal column chromatography to remove contaminating small molecular weight DNA and RNA oligonucleotides. These non-specific oligonucleotides can be used as primers in subsequent mutagenesis reactions, causing false-positive phage spots in the background.
16. Measure the DNA at 260 nm ( 1OD260 = 40ug/ml) using a spectrophotometer (see Appendix 8, DNA Quantification). Using the initial single-stranded M13 recombinant DNA as a molecular weight standard, take 0.5ug of the above DNA sample and analyze the DNA molecular weight size by gel electrophoresis.
17. Perform oligonucleotide directed mutagenesis analysis as described in Scheme 2.