We have mixed genotype litters of pups that we would like to culture, but it is difficult to do the genotyping before culturing the microglia (either E19 or P0 pups) and we were wondering how other people best get around this issue. We could use really small flasks, but the scale-up-down effects are typically not linear in cell culture which also makes me nervous. Thanks for your advice!
Are you trying any rapid genotyping methods? We use HOTSHOT method for quick genomic DNA extraction from tail samples, which generally takes only 20 minutes. The genotyping takes another 1 hr 20 min or so, plus another 15 min to run the gel. In total you should be able to get the results in less than 2 hours. If you try a Phusion based enzyme you should be able to speed up the PCR and cut the time in half. Some enzymes have been designed to work well with tissue used directly in the PCR reaction without prior DNA extraction.
I should also mention that I have done culture of primary neurons from rat embryos and I have at times needed to keep the string of embryos on ice for nearly 2 hours (after removal from the euthanized mom) and was still able to get perfectly healthy neurons. I assume the same may be possible for microglia to stall for more time while waiting for the genotyping results...although the embryos would have to be removed completely to get the tissue samples.
Thanks ! We have not looked at the fast genotyping methods, but that might be the best alternative as I know that they can be assessed using a melt curve analysis on a RT machine. Perhaps also easer in the long run than handling multiple flasks. :-)
Have you had any experience with the melt curve type analysis?
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