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Flox and Cre Primers - PCR Troubleshooting


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#1 Superman

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Posted 24 October 2009 - 02:44 PM

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


#2 Mighty Mouse

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Posted 25 October 2009 - 10:37 AM

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.
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#3 Superman

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Posted 26 October 2009 - 11:04 AM

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




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