positive colony PCR, but no insert!? - (Apr/10/2008 )

Hi,
Just found out by sequencing that my pCMV-script is empty, but I did got positive results by colony PCR. I'm a bit confused now.....

I did colony PCR on 7 clones,
2 was negative, 2 was positive with a nice single band, and 3 was 'even more positive' with smear and an extra band.
So I guess it could not be contamination of reagents since I have negative clones as well..

I did a maxiprep on one of the 'single band' positive.

Can a vector 'kick-out' the insert?

If someone has the time to explain how this has happened, I'll be so greatful :-)

If your primers bind only to the insert, then you can get amplification from DNA left over from the ligation mix, into the transformation, and spread on the plate. Very small numbers of molecules are enough. Ideally you want a primer pair that primes once on the insert and once on the vector (not that I always do that).

Thanks Phage!!!

That makes sense :-)
From now on I'll be careful with trusting the colonyPCR and maybe choose another primer.

Hey,
I gave up on PCR screeing, I go with restriction digestion all the time. PCR gives positive when you digest you find nothing.

I would confirm with digestion for sure and trust it more than PCR

Well, of course digestion will give you better confirmation than PCR colony but it has its advantage of time saving. Usually it works like 90%. At least for me.

Even with digestion, you still need to send for sequencing, that is for sure.