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Salient

Salient is an excellent design with a fresh approach for the ever-changing Web. Integrated with Gantry 5, it is infinitely customizable, incredibly powerful, and remarkably simple.

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Site directed mutagenesis using KOD polymerase

  1. Design primer with the mutation in the middle and ~15 bases on either side. Order complementary primers on both strands. Dissolve at 10 pmol/µl.
  2. Prepare PCR mixture
    • 10 ng plasmid DNA
    • 1.25 µl each primer
    • 5 µl 10x KOD buffer
    • 5 µl 2mM dNTPs
    • 3 µl MgSO4
    • 1 µl KOD polymerase
    • H2O to 50 µl
  3. Run 12 cycli of PCR:
    • 95°C - 2 min
      • 95°C - 20 sec
      • 55°C - 10 sec
      • 70°C - 5 min
    • 70°C - 5 min
    • 4°C - 5 min
  4. Add 1 µl DpnI (to digest methylated DNA)
  5. Incubate for 1.5 hrs at 37°C
  6. Transform into bacteria