Single-Stranded DNA Isolation and Mutagenesis
Getting ssDNA
- Transform plasmid into dut-ung-FÕ cells (CJ236) on LB/Ab selection plate.
- Inoculate a single colony into 2X TY media + 100 μg/μl amp + 0.1% glucose. Grow O/N 37°C.
- Take 100 μl of O/N culture and add 3 μl M13K07 helper phage and incubate 20 minutes 37°C.
- Add 3 ml 2X TY + 100 μg/ml amp + 20 μg/ml kanamycin + 0.25 μg/ml uridine + 0.1% glucose. Incubate at 30°C for 20-24 hours or 37°C for 8-12 hours.
Some researchers take colony into this, plus phage, and incubate 37°C.
For 3 ml, add 6 μl 50 mg/ml amp, 6 μl 10 mg/ml kan, 0.3 μl 2.5 mg/ml uridine, 30 μl 10% glucose. - Transfer to Eppendorf tubes and spin bacteria down at 12K for 2 minutes. Save supernatant (phage).
- Add 1/4 volume 20% PEG-8000/3.5M ammonium acetate. Incubate at RT for 15 minutes.
- Harvest phage by spinning at 12K at 4°C for 20 minutes. Thoroughly drain supernatant.
You should have a white pellet at this point. - Resuspend pellet in 1/10 volume of fresh digestion buffer*.
- Heat at 55°C for 20 minutes.
- Extract DNA 1X with phenol:chloroform, then 1X with chloroform.
- Precipitate DNA with 1/10 volume 3M sodium acetate, then with 2 volumes ethanol.
- Dissolve DNA in 20 μl H2O, and check 1 μl on gel. ssDNA with uracil incorporation should run similarly to undigested plasmid DNA -- it is about the same size.
*Digestion Buffer
- 1mM EDTA
- 10mM Tris, pH 8.0
- 0.2% Sarkosyl
- 50 μg/ml Proteinase K
1 ml
- 965.5 μl H2O
- 2 μl 0.5M EDTA
- 10 μl 1M Tris
- 20 μl 10% Sarkosyl
- 2.5 μl 20 mg/ml Proteinase K
Mutagenesis
Phosphorylation of Oligonucleotides
- Set up phosphorylation reaction**.
- Incubate at 37°C for 30-45 minutes.
- Heat-inactivate kinase at 65°C for 10 minutes.
**Phosphorylation Reaction
- 20 pmol Mutagenizing Oligo
- 2 μl 10mM dATP
- 4 μl 5x Kinase Buffer
- 1 μl T4 Polynucleotide Kinase (NEB)
- 12 μl H20
- Total volume: 20 μl
Annealing
- Set up annealing reaction†.
Run one tube with no oligo, as a control. - Heat at 70°C for 5 minutes in a beaker of water in hybridization oven.
- Take beaker of water out on benchtop with thermometer and let cool gradually to 30°C (~40 minutes to 1 hour).
†Annealing Reaction
- 1.2 μl ssDNA (50-100 ng)
- 7 μl Phosphorylated Oligo (7 pmol)
- 1 μl 10x Annealing Buffer
- 0.8 μl H2O
- Total Volume: 20 μl
10x Annealing buffer
- 200mM Tris, pH 7.4
- 20mM MgCl2
- 500mM NaCl
Second Strand Synthesis
- Set up synthesis reaction‡.
- Incubate for 5 minutes at RT.
- Incubate for 90 minutes to 3 hours at 37°C
- Run 5 μl on gel.
You should see an upward shift compared to ssDNA.
‡Synthesis Reaction
- 1 μl 10x Synthesis Buffer
- 1 μl 10mM dATP
- 0.2 μl 0.1M DTT
- 0.5 μl T4 DNA ligase
- 0.5 μl T4 DNA polymerase
10x Synthesis Buffer
- 175mM Tris, pH 7.4
- 37.5mM MgCl2
- 4mM dNTPs
Transformation
- For heat-competent XL-1 blues, use 7 μl of reaction.
- Plate out on LB/Amp.
- Expect significantly fewer colonies on mutated plates compared to control.