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

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PCR Multiple Mutation PCR: Two 50 μL reaktions with all primers and one PCR control with only the flanking primers: 5 μL 10x Taq ligase buffer 1 μL GFPrv (10 pmol/μL) 1 μL GFPfw (10 pmol/μL) 2.5 μL dNTP mix (4 mM each) 1 μL Pfu (= 3u; Promega) 0.5 μL Taq ligase (= 20u; NEB) x μL plasmid pBAD-GFP (100 ng) 1 μL of each mutagenic primer (GFPmut1fw, GFPmut2rv, GFPmut3fw, GFPmut4rv, GFPmut5fw, GFPmut6fw) x μL A. dest Program: 5 min at 95°C 35 cycles: 30 s 95 °C 30 s 57°C 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.