Damien Marsic wrote:
>> I have trouble doing PCR with whole bacteria and I would like to receive
> some advice.
> I want to screen colonies from transformed bacteria by PCR. I use T3 and T7
> primers, since the plasmid used has T3 and T7 priming sites flanking the
> cloning region. Knowing the distance between these sites in the original
> plasmid, the size of the PCR products will tell me if there is an insert or
> The problem is that I can't succeed in doing the PCR (although it worked
> fine when I did it a few months ago with the same plasmid and the same
> bacterial strain). From 3 experiments involving a total of 50 samples, the
> PCR worked in only 6 of them. For the 44 others, there was no amplification
> at all. I used Pfu and Tfl polymerases, in 50 ul reactions, with 5 to 10 min
> initial denaturation at 95 deg C (to lyse the cells and inactivate
> nucleases) followed by 30 cycles with standard parameters.
> If you have some experience or a efficient protocol, please let me know.
> Please always copy me your message to marsicd at email.uah.edu>> Damien
I don´t know the distance between the T3 and T7 priming sites in your vector
(without insert), but isn´t it possible that you have only 6 positive clones
(and 44 negative ones, like religated plasmid). Probably the PCR product of the
religated vector is too small to see on the gel (or to tell it from the excess
primers that run in the same region of the gel if you use a standard agarose
Or, your "standard parameters" are not working properly for that particular
PCR, most probably because of a too short extension time. Use 2min per kb for
Pfu polymerase, or, even better for this kind of analysis, use Taq. Taq is much
more robust (although not as heat stable) than Pfu, and much cheaper too.
And what about doing standard minipreps to identify positive clones? It is much
faster than trying PCR several times until it works, and even cheaper than PCR
with Taq (if you don´t use a kit for minipreps, of course...)
Anyway, 6 clones out of 50 colonies is not too bad a result for a difficult
cloning. Be happy and proceed with analysis...