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Single-Stranded DNA Isolation and Mutagenesis

Getting ssDNA

  1. Transform plasmid into dut-ung-FÕ cells (CJ236) on LB/Ab selection plate.
  2. Inoculate a single colony into 2X TY media + 100 μg/μl amp + 0.1% glucose. Grow O/N 37°C.
  3. Take 100 μl of O/N culture and add 3 μl M13K07 helper phage and incubate 20 minutes 37°C.
  4. 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.
  5. Transfer to Eppendorf tubes and spin bacteria down at 12K for 2 minutes. Save supernatant (phage).
  6. Add 1/4 volume 20% PEG-8000/3.5M ammonium acetate. Incubate at RT for 15 minutes.
  7. Harvest phage by spinning at 12K at 4°C for 20 minutes. Thoroughly drain supernatant.

    You should have a white pellet at this point.

  8. Resuspend pellet in 1/10 volume of fresh digestion buffer*.
  9. Heat at 55°C for 20 minutes.
  10. Extract DNA 1X with phenol:chloroform, then 1X with chloroform.
  11. Precipitate DNA with 1/10 volume 3M sodium acetate, then with 2 volumes ethanol.
  12. 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

  1. Set up phosphorylation reaction**.
  2. Incubate at 37°C for 30-45 minutes.
  3. 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

  1. Set up annealing reaction†.

    Run one tube with no oligo, as a control.

  2. Heat at 70°C for 5 minutes in a beaker of water in hybridization oven.
  3. 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

  1. Set up synthesis reaction‡.
  2. Incubate for 5 minutes at RT.
  3. Incubate for 90 minutes to 3 hours at 37°C
  4. 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

  1. For heat-competent XL-1 blues, use 7 μl of reaction.
  2. Plate out on LB/Amp.
  3. Expect significantly fewer colonies on mutated plates compared to control.

© 2014 The University of Texas MD Anderson Cancer Center