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This protocol is now superseded - UPDATE SOON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):

-> 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:

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):

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.

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