Single Cell PCR
(Protocol provided by Carolyn Troeger)
- Cell picking c Axiovert 100/Zeiss, extended glass capillary/Drummond, Broomall and a micromanipulator/Zeiss
- 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)
- Overlay c mineral oil if needed (depending on PCR machine) and incubate @ 50°C for 1 h, denaturate @ 99°C for 30 min.
- 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.
- 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.
- Run products on 2% Agarose gel