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 µl of 400 ng/µl Proteinase K/17 µM SDS (50 µl stock = 1 µl 20 mg/ml Prot K + 49 µl 0.005% SDS)
3. Overlay c mineral oil if needed (depending on PCR machine) and incubate @ 50°C for 1 h, denaturate @ 99°C for 30 min.
4. Keep tube c cell on ice. Add 40 µl 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 µl aliquots of the external PCR product is placed into a new PCR tube containing 20 µl 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