Same-Day Genotyping of Isolated Gastrulating Murine Embryos

After isolating the visceral yolk sac from the mouse embryo, proceed to digest each yolk sac in an eight-strip polymerase chain reaction or PCR tube. Using a P20 pipette, add 19.3 microliters of PCR template DNA lysis buffer, and 0.7 microliters of 0.2 micrograms per milliliter proteinase K to each yolk sac sample. Vortex the sample for 10 seconds.

And using a mini centrifuge with a strip adapter, spin down the samples at 1000 G for 10 seconds. Place the eight-strip PCR tube in an 85 degree Celsius heat block for 45 minutes while vortexing for five seconds every five minutes. After 45 minutes, spin the tube strip down again at 1000 G for 10 seconds before proceeding with PCR for desired genetic identification.

To perform the PCR reaction for Cre genotyping, prepare a PCR master mix containing the displayed components per eight strip PCR tube for each yolk sac. Proceed to run the PCR thermal cycle amplification program using the displayed cycle. Then run the PCR products on a 1%agarose gel to draw genotyping conclusions.

Store the remaining digested yolk sac samples in a minus 20 degree Celsius freezer for long-term storage. When the PCR product was separated on an agarose gel, expected fragment sizes for the LoxP and wild type alleles, as well as the Cre allele were seen.