This protocol is now superseded - UPDATE SOON
With credit to Bentley Fane and Maniartis.
Step 1: Primer phosphorylation
We phosphorylate 200pmol primer (Fane carries this out in 2ul reaction mixture):
- 2ul 100uM primer
- 2ul 10mM ATP
- 2ul T4 ligase buffer
- 1ul 10U/ul T4 polynucleotide kinase
- 13ul water
-> 37oC for 30 minutes.
Step 2: Relax secondary structure and anneal
We add 10pmol of phosphorylated primer to 0.5pmol ssDNA in ligase buffer (Fane uses PE1 buffer). Start boiling water in a bowl using tripod arrangement. Meanwhile to each tube add:
- 2ul ssDNA diluted 1/100, 1/10, or 1 times (SHOULD PURIFY BAND)
- 1ul phosphorylated primer mix (above)
- 6ul water
- 1ul T4 ligase buffer
When water is boiled, remove bowl from heat and immerse tubes in it. Store bowl (with tubes) in cold room or fridge (+4oC) for 40 minutes to 1 hour.
Step 3: Non-gapped extension reaction
We add ATP to 1mM, dNTPs to 0.5mM, 4 Weiss Units ligase, 2U polymerase in ligase buffer to 20ul (Fane uses PE1 buffer). The following is added to each tube (error here unless 10ul in previous):
- 1ul ligase buffer
- 2ul 10mM ATP
- 1ul 2mM (each) dNTPs
- 3.3ul water
- 0.7ul 3U/ul T4 DNA polymerase
- 2ul 2U/ul (400 c.e.u.s) T4 DNA ligase
Reaction kept on ice for 5 minutes, room temperature for 5 minutes, then two hours at 37oC. After which the reaction may be frozen.
Step 4: Prepare for transformation
Fane uses 2ul of 1/10 dilution of this for transformation into 100ul competent cells. Transformation efficiency improved by O/N storage.
From Fane: We incubate the transformation mixtures for 30 minutes on ice, heat shock for 1 minute at 42oC, then ice again for 5 minutes, and then we plate. This typically gives us 200 plaques per plate.
The repair can go either way in the cells, so you will need a way to either directly select for your mutant, or an efficient way to screen the plaques by toothpicking into indicator lawns.