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RESEARCH DIVISION Laboratory Manual

 


 

PCR Genotyping from the Embryo Yoke Sac

This method is modified from Dr. Lyn Corcoran (WEHI)

DNA buffer :

1 X PCR buffer
0.45% NP - 40
0.45% Tween - 20
Proteinase K 100 mg / ml

Prepare the DNA for PCR :

  1. Add 50l DNA buffer to a PCR tube
  2. Dig washed yolk sac into tube and spin down
  3. Incubate at 50C for 30 minutes,
  4. Boil the lysate for 5 minutes, spin down,
  5. Take 5l for PCR.

PCR reaction (50 l) contains :

1 X PCR buffer
2 mM MgCl2
100 nM dNTPs

Primers :

a. 0.25 M
b. 0.05 M
c. 0.2 M

a________c     400bp (targeting region)

a________b     200bp (wild type)

2.5 unit/ 100ulTaq DNA polymerase

PCR cycle :

Initial denaturing by 94C for 4 minutes
28 cycles of 94C for 1 minute
60C for 1 minute
72C for 1 minute
Final extension by 72C for 10 minutes
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998