Team:Washington/Notebook/SOEingPCR

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SOEing PCR

  1. Design primers
    • VF2 / VR = standard forward and revers primer from original construct
    1. For mutations/insertions/deletions of 1-15 base pairs
    • Forward: 5'-----------------------XXXX-----------------3'
    • Template: 5'----------------------------------------------3'
    • Reverse: 3'-----------------------XXXX-----------------5'
      • X = mismatch base pair with template
      • - = Matching base pair with template. Make sure that 15-25 base pairs match. The Tm of the matching regions should be >60C, and the GC content 40%-60%
    1. For mutations/insertions/deletions of 15+ base pairs
    • Forward: ..............................................5'-XXXX-----------------3'
    • Template:..5'------------------------------------------------3'
    • Reverse:...3'-----------------------XXXX-5'
      • X = mismatch base pair with template
      • - = Matching base pair with template. Make sure that 15-25 base pairs match. The Tm of the matching regions should be >60C, and the GC content 40%-60%
  1. PCR: Adding the mutations
    • An example assembly altering DNA at three different points (can do this with as many or as few as needed for your application)
      • Top Strand..... T7F........M1-F..................M2-F....................M3-F...................
      • Ref Sequence ...................X.........................X...........................X.....................
      • Bottom Strand ...............M1-R..................M2-R....................M3-R............T7R
      • MX-F = forward mutation primer X
      • MX-R = reverse mutation primer x
      • X = mutation site
      • ..... = place holder
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