I need to genotype single 27hpf zebrafish embryos that have already undergone WISH but I'm not sure how is best to extract the DNA given that they have previously been fixed in 4% PFA and are now stored in glycerol.
Thanks in advance for any advice!
Hi Yasmin,
Attached is a protocol I used for genotyping zebrafish after a number of different stainings and stages. All you need is a bit of tissue and you might have to wash the embryos in PBS to remove the glycerol. The embryos being fixed is no problem.
The protocol was adapted from the ZIRC genotyping protocol. It is basically the same protocol as is used for genotyping life fish.
https://zebrafish.org/documents/protocols/pdf/Genotyping/ZIRC_Genotyping_A_H_Combined.pdf
Good luck with your experiments
Best Simone
Hi Simone,
Thank-you for sending me the protocols. Have you found whether different stains or staining times etc. affect the level of DNA recovery?
Best wishes,
Yasmin
Hi Yasmin,
The duration of staining and the type of staining should not affect the DNA recovery. We regularly genotype embryos after colorimetric and fluorescent in situ hybrization as well as other types of stains without a problem.
Best
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