User:Torsten Waldminghaus/Notebook/Multiple Mutation Reaction/2008/04/28

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PCR
Two 50 μL reaktions with all primers and one PCR control with only the flanking primers:
 * Multiple Mutation PCR:


 * 1) 5 μL 10x Taq ligase buffer
 * 2) 1 μL GFPrv (10 pmol/μL)
 * 3) 1 μL GFPfw (10 pmol/μL)
 * 4) 2.5 μL dNTP mix (4 mM each)
 * 5) 1 μL Pfu (= 3u; Promega)
 * 6) 0.5 μL Taq ligase (= 20u; NEB)
 * 7) x μL plasmid pBAD-GFP (100 ng)
 * 8) 1 μL of each mutagenic primer (GFPmut1fw, GFPmut2rv, GFPmut3fw, GFPmut4rv, GFPmut5fw, GFPmut6fw)
 * 9) x μL A. dest

Program:

5 min at 95°C

35 cycles:
 * 1) 30 s 95 °C
 * 2) 30 s 57°C
 * 3) 5 min 65°C

Note: we have a quite old PCR machine and I had problems programming it so that the samples incubated at 65 °C for about 2 hours. I than started the right program and left it over night at 4 °C in the machine.