Herskowitz Lab Protocol
From : Anita Sil


PCR to Check Genotype

  1. pick one colony, r/s in 100ul PCR reaction mix (use new toothpicks), use "one toothpick full - solution should be cloudy on r/s"
  2. boil 5' and spin briefly
  3. add 0.5ul Taq Polymerase (2.5U) and overlay reaction with oil [also works with Boerhinger Expand Polymerase mix]
  4. amplify 30 cycles :
    • 94°C - 1'
    • 42°C - 2'
    • 65°C - 4'
  5. Temperatures are critical
  6. do final extension at 65°C for 5'
  7. remove 10-15ml and check on an agarose gel

PCR Reaction Mix

  • 63.5ml water
  • 10ml 10X PCR buffer [100mM Tris 8.3, 500mM KCl, 15mM MgCl2]
  • 16ml 1.25mM each dNTP
  • 5ml - each primer 2mM conc

-- final vol = 99.5 ml


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