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:

  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.


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