I want to perform western blotting in mouse embryos while knowing their genotype. I want to collect as much sample as possible for western blotting, and do it as quickly as possible after dissection. In stead of splitting the fresh embryo into two parts for western blotting and genotyping at the beginning, can I treat the whole embryo following western blotting sample preparation procedure, and then take out a little sample from that embryo for genotyping? Below is my brief procedure:
Freeze the fresh embryos by liquid nitrogen. When ready, thaw the embryos and put them in homogenization buffer, and homogenate samples. Take several microlitters for genotyping, and the rest for western blotting.
I'm not sure if the genomic DNA can survive the homogenization buffer before genotyping. My homogenization buffer contains sucrose, EDTA, EGTA, HEPES, SDS, protease inhibitor, and protein phosphatase inhibitor.
Any comments or alternative strategies are welcome! Thank you!
Unless your embryos are very early stage you should have enough material to isolate DNA and protein separately, which is the best option. It might be possible to use a product like quickextract to recover some DNA after it has been in your lysis buffer.
Thank you Mark for your comment.
The embryo I'm working on is E9.5-10.5. I'll first try to scrape just a little bit tissue from the embryo for genotyping before it is subjected to western blotting.
Hi Mark, the E9.5 /10.5 (from mouse) is very small as I observed. I may end up trying the alternative way to recover DNA from tissue lysate. Is the product, QuickExtract™ DNA Extraction Solution from epicentre, you recommended? Thank you.
http://www.epibio.com/applications/nucleic-acid-purification-extraction-kits/dna-extraction/quickextract-dna-extraction-solution?details
Hi Hongtian
Did your genomic DNA survive the homogenization buffer? or you tried someother technique? Please let me know what you did then to do genotyping?
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