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