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RESEARCH DIVISION Laboratory Manual

 


 

PCR Genotyping of Embryos and Blastocysts

These protocols have come from the transgenic mouse list and are intended as a guide, none of them have been tried in this lab

Cell(s) transferred in small volume of water to a .5 ml tube and lysed by rapid freeze / thaw. Buffer was then added to the lysate before the sample was further processed for RT-PCR, though I don't know why it wouldn't be effective for regular PCR.

Ref: Biotechniques 24(4): 618-23.1998.

One protocol called for simply heating the cell(s) in water at 94C for a few minutes then diluting some of the sample in a standard PCR rxn mixture. The really interesting part about this paper is it describes a method for embryo biopsy in which a single blastomere is removed for PCR and the remaining embryo is returned, after incubating O/N while PCR was being performed, via uterine transfer to pseudopregnant moms, thus resulting in viable pre-selected mice. This protocol sounds relatively simple in that anyone with experience in transgenic techniques could probably do it.

Ref: PNAS 87: 4053-4057, 1990.

The vast majority of responses to my query were in favor of the following protocol:

Cell(s) are transferred in a small volume to a .5 ml tube to which PCR buffer containing 0.45% NP-40, 0.45% Tween-20, and proteinase K (PK) is added. The final concentration of PK being anywhere from 60-400 ug/ml. This is then heated to anywhere from 50-65C for 30-60 min. then the sample is heated to 95C or boiled to inactivate the PK. A small volume of this lysate is then processed normally for PCR.

Ref: Transgenic Res. 4(1):12-17, 1995,

There are also several variations on this:
  1. PCR buffer with DTT, SDS, and PK. Nature 335(29): 414-417,1988.
  2. PCR buffer with Guanidine HCL and PK. Methods in Enzymology 225: 557-583, 1993.
  3. Alkaline lysis with KOH and DTT. PNAS 86: 9389-9393, 1989.

These protocols were used to PCR DNA from Embryos, yolk sacs, blastocysts, blastomeres, single sperm, and even cell scrapings from ICM off of frozen sections.

 

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