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Single Cell PCR

Single Cell PCR

(Protocol provided by Carolyn Troeger)

  1. Cell picking c Axiovert 100/Zeiss, extended glass capillary/Drummond, Broomall and a micromanipulator/Zeiss
  2. Transfer cell into PCR tube containing 5 l of 400 ng/l Proteinase K/17 M SDS (50 l stock = 1 l 20 mg/ml Prot K + 49 l 0.005% SDS)
  3. Overlay c mineral oil if needed (depending on PCR machine) and incubate @ 50C for 1 h, denaturate @ 99C for 30 min.
  4. Keep tube c cell on ice. Add 40 l of PCR mix containing 25 pmol each external primer, 1.5 mmmol/l MgCl2, 300 nm0l/l dNTPs and 2 IU Taq. Amplify.
  5. 1 l aliquots of the external PCR product is placed into a new PCR tube containing 20 l of PCR mix c 10 pmol of each internal primer and MgCl2, dNTPs and Taq as above. Amplify.
  6. Run products on 2% Agarose gel