genotyping mice-no pcr product - (Feb/17/2006 )
I have been genotyping mice with a protocol given by the lab who gave us the mice. there are 2 sets of primers in the pcr for +/+ and +/- so there will be a product from each pcr reaction.
I extracted DNA from tails by proteinase K treatments and isopropanol, spooled out the DNA, dissolved in TE and PCRd.
I made up a master mix for 17 samples, 15 gave pcr products and 2 others gave nothing...
I repeated the pcr, still nothing.
I repeated the tail snip, DNA extraction and pcr, and one is still not giving a pcr product.
There was a lot of DNA that I spooled out, and it completely dissolved in the TE before I made up the PCR reactions,
do you think I could have something in the DNA that is preventing the PCR from working ?
am thinking of re-precipitating the DNA and repeating the PCR, any ideas ?
thanks for your help
Why not use Tris or ddH20 instead of Tris-EDTA?
It's possible that the EDTA is giving you problems.
had been thinking about the EDTA (its 0.1mM), but surely that would be a problem with all the samples, so far only 1 out of 17 hasn't worked...
will re-precipitate and resuspend without EDTA
Ah, now I read more carefully.
I would look at the amplicon to see if there are any GC rich regions. If that is the case, I would try supplementing with 3-5% DMSO or 1.0 M Betaine or both.
I would also try a gradient for the annealing temperature. Sometimes higher annealing temperatures are required to remove deleterious secondary structures.