Hello everyone,
I'm facing problems with getting an intact genomic DNA from zebrafish embryos. I have tried to use couple of protocols given in ZFIN network website, but do not see one single band when I try to run the extracted sample on 1% native agarose gel. I'm using the below protocols, the downstream applications is PCR and then digestion of PCR products. If anybody see any issues with what I'm using/doing, please help me in pointing it out. Or if anyone has any other protocol which works, please share it with me.
Dechorionated embryos in microfuge tubes without any left over liquid from the medium incubated with Extraction buffer (10mM Tris pH 8.2, 10mM EDTA, 200mM NaCl, 0.5% SDS, 200 ug/ml proteinase K) for 3 hours at 50 C in a thermal block. I add 100 ul of extraction buffer for 10 pooled embryos.
After incubation, add 200 ul of 100% EtOH, mix and place on ice for 20-30 mins, centrifuge for 10 min, remove supernatant and add 200 ul 70% EtOH. Spin again for 2 min, remove liquid and dry pellet, resuspend in water.
Thanks
Sindhu
Hi based on your application, crude gDNA is ok. We just add 3ul 50mM NaOH per embryo and heat at 95 degree for 15 min, cool to RT and then balance with 1/10 volume of 1M Tris-HCl(pH8.0) . It works perfectly for PCR.
Zebrafish has 25 pairs of chromosomes, so visualising a single band is not a sign of intact DNA but limitation of your analysis method. If you see about 25 bands - this should be about right, however it is impossible to separate them on 1% gel. If you don't see any DNA and you have a pellet, please make sure the pellet is dissolved (it can be difficult).
Hello, Thank you for your response. I expected one single band due to images like below given by some kits which are commercially available to extract genomic DNA from tissues and whole embryos. In comparison, my gel picture looks bad but if you say more bands are all right, then I will try to go ahead with PCR to see if I get right sized bands amplified.
If you have a band which is higher Mw than your top marker, this are all chromosomes together. The bands below are not chromosomes. Your PCR should work, just don't overload with gDNA. Dilute 1 ul DNA in 1000 ul water and use 2ul of this solution as a template per reaction, otherwise you will inhibit the PCR.
Best regards
Hi based on your application, crude gDNA is ok. We just add 3ul 50mM NaOH per embryo and heat at 95 degree for 15 min, cool to RT and then balance with 1/10 volume of 1M Tris-HCl(pH8.0) . It works perfectly for PCR.
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