Flox and Cre Primers - PCR Troubleshooting - Please Help (Oct/24/2009 )

Hi everyone,

I am having a PCR nightmare at the moment. My PI has been doing this exact PCR with the same reagents even a few weeks before me and it was working fine.

I am currently genotyping some Flox and Cre mice. For some reason the Flox bands are coming out great but the Cre ones keep failing to work. I have changed Primers, Water, Tubes, All reagents etc. What can I do? Perhaps a Temperature gradient? How would I do that? It can't be the primers or the systems as they have been done over and over again! Any ideas anyone?

The program I am using is as follows:

95 degrees for 5 min

40 cycles of:
95 for 20s
55 for 60s
72 for 60s
end cycles

final extension 5 min
hold at 5 degrees

I have tried two different systems with different taqs

Crimson Taq:

5 x Crimson Buffer - 5ul
dNTP (10um) - 0.5ul
Primer 5' - 0.5ul
Primer 3' - 0.5ul
Taq - 0.125ul
DNA - 1 or 2ul
Water - Make up to 25ul

Other Taq:

10 x MGB Buffer - 2.5ul
DMSO - 2.5ul
dNTP - 2.5ul
Beta Mercaptoethanol - 2.5ul
Primer 5' - 0.2ul
Primer 3' - 0.2ul
Taq - 0.2ul
DNA - 1ul
Water - Make up to 25ul

I've done some genotyping of some flox/Cre mice; unlike genotyping of some of the other mice in our lab I find that the primers "go bad" faster for this set than others. Have you tried re-ordering your primers? Making sure they are the right concentration etc? Or perhaps there is an issue with your positive control for the Cre? I've always found end-point PCR to be pretty robust, I'm surprised you're not seeing anything.

Thanks for your post.

One of the Cre Tm's is 52 while the other is 59 so they are not the best.

I did a temperature gradient PCR today and it worked at 52 degrees. I did re-order the primers, so I guess that's the key. I am so happy it finally worked after 5-6 PCR's.

Cheers,
Superman