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Problem with PCR on bacmids

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#1 mgn67



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Posted 22 September 2010 - 04:31 AM

I'm trying to perform PCR on a bacmid i purified from DH10Bac bacterias, to confirm the good transposition of my gene in the bacmid. My problem is that the PCR doesn't seem to work... I've tried several times, from several minipreps, with "gene" forward / M13 reverse primers, or with M13 forward/reverse primers, but nothing work. I could have thought the problem was coming from my primers, or the enzyme, or i dunno what, but other people are using the same stock, to perform the same kind of PCR on bacmids, and it works well for them ! Though i'm using the same protocol, same volumes, etc...
The thing that is weird, is that even with M13 forward/reverse primers, i don't get anything but a band very low on the gel corresponding to the primers themselves. Not even a 300bp band which could at least mean the recombination didn't work...

Any idea ? I'm in dispear !

Thks, Morgan.

#2 phage434



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Posted 22 September 2010 - 05:01 AM

The usual problem is too many cells in the PCR reaction. Dilute by picking a colony on a 10 ul tip, putting it into 50 ul of water, then spotting 2 ul on a master plate. Vortex the 50 ul dilution, and use 0.5 ul in a 10 ul PCR reaction. If you have a QPCR machine, you can identify positives without even waiting for it to complete.

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