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Whole Genomic DNA and when to cut

Genomic DNA restriction enzyme PCR Genotyping

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#1 xinobi

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Posted 28 February 2013 - 04:49 PM

Hi, I recently had to do a lot of genotyping for the transgenic mice in our lab. It was very easy, just cut the tails, extract the DNA, run the PCR, and visualize with a gel. My question is, I previously remember having to "genotype transgenic mice" before, and was required to do a restriction digest. I can't for the life of me remember why, if I can just extract whole genomic DNA, and be able to genotype accurately with that. I remember my PI yelling at me like a dunce because I wanted to use whole genomic DNA then go straight to PCR. Is there a reason I could have needed to do a restriction digest? Moreso to the point, once DNA is extracted it remains whole genomic DNA, like.. in a circle, shouldn't I have to somehow cut it in order for the PCR denaturing step to come along and cleave the double stranded DNA lengths? And also generate workable, straight lengths of DNA to work with? Or does the denaturing step in PCR do enough to separate the double strands for the primers to come along and amplify where they need to? Thanks!!

#2 bob1

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Posted 28 February 2013 - 11:55 PM

I think you misunderstand eukaryotic DNA - most of it is more or less linear, not circular like plasmids. In general genomic DNA should be fine for PCR - are you sure the "restriction digest" you were doing wasn't a RNase step? Otherwise there may be some specific insert in the DNA that when combined with the PCR gives you the genotype. However, this would be particular to the exact mouse strain you are using.

#3 Steven Creacy

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Posted 01 March 2013 - 06:52 AM

Your PI should have given you a clear, written protocol for the genotyping and interpretation of results. Every engineered mouse strain is different. Some require restriction digest before or after PCR, because of interference by pseudogenes or other complications. Your protocol would tell you which enzyme to use- there are hundreds. Just getting a band on a gel is not necessarily enough- the hand size is used to score the genotype and often the zygosity. If you have a lot of mice to deal with, your PI should consider an automated gentoyping service. It saves money in time, cage costs and peace of mind regarding the use of the animals in expensive experiments; and some offer a free trial.

Edited by Steven Creacy, 01 March 2013 - 07:26 AM.






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