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-   -   Mouse Tail Genotyping Problems (http://www.molecularstation.com/forum/dna-techniques/88094-mouse-tail-genotyping-problems.html)

dkg87 09-13-2013 05:29 PM

Mouse Tail Genotyping Problems
 
I am attempting to genotype my mice for a transgene that will express an shRNA (gene knockdown technique). I have been doing this screen successfully for months using a certain protocol. Recently however it has stopped working for no apparent reason. I have tried new primers, buffers, dNTPs, and taq and still no luck. I am currently doing a gradient PCR to see it will work at a new annealing temperature. Does anyone have any other ideas???

Thanks

DKG

luisillo 09-17-2013 02:34 PM

Re: Mouse Tail Genotyping Problems
 
Check if your getting nucleic acid from your extraction, the problem might be there. Don't you have an internal control? I mean, a gene that should always amplify regardless your problem gene (i.e. b-actin for human assays; do not know which might work well in mice, though)

dkg87 09-18-2013 02:01 PM

Re: Mouse Tail Genotyping Problems
 
I have quantified the DNA it is "pure" based off the 260/280 and 260/230. I have also used the same DNA for other screens and it's worked fine. We don't usually use an internal control, but should probably start. It doesn't make sense to me that these primers and buffers were working fine a few months ago and now all of a sudden stop.

Thanks again

DKG


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