4 replies to this topic

[imladris]

I have been attempting to use colony PCR to screen colonies for a bacmid carrying a gene of interest. (I electroporated DH10Bac cells carrying helper plasmid (transposase) with plasmid containing GOI between Tn7 sequences).

However, I found that using the forward and reverse M13 primers resulted in a product of expected size (~5.2 kb...long PCR reaction!), but using a gene-specific primer and the appropriate M13 primer did not result in product (<2 kb products). I tried two different gene-specific primers with the forward and reverse M13 primers for each, to ensure that there was no issue with opposite orientation. I had previously used the gene-specific primers in PCR with no issues, and I am confident that my reaction conditions are appropriate, based on previous colony PCRs, and previous use of the gene-specific primers. I thought that the 'no PCR product' result could be attributed possibly to using too much colony, but even after reducing the amount, there was no amplification with the gene-specific primer/M13 primer combinations, while I still had amplification with the two M13 primers.

I then decided to directly purify bacmid from colonies that were positive by colony PCR with the two M13 primers. I then used the purified bacmid as a template (after dilution) for PCR with the previously negative gene-specific primer/M13 primer PCRs. I had no product, but there was the previously observed expected product with the two M13 primers. I also did PCR with insert-specific primers, to verify that the GOI was present in the purified samples. Of course, that  did not verify that the GOI was in the bacmid...the original plasmid may also have been purified.

I then used the PCR product from the M13 primer PCR as a template in a PCR with the gene-specific primer/M13 primer combinations that were previously negative. I had the expected product.

Can anyone help to explain these results, and perhaps suggest what may be the cause of colony PCR being consistently negative with a gene-specific/M13 primer combination?

Thank-you!

[nightingale]

Hello imladris,

will you please clarify this sentence more ???

Quote

I then used the PCR product from the M13 primer PCR as a template in a PCR with the gene-specific primer/M13 primer combinations that were previously negative. I had the expected product.

and have you checked the positive product of the general M13 primers, for the specific primers of your interest ???

[Trof]

AFAIK she used the M13 product for a nested PCR using the gene-specific primer.

If you are getting the nested PCR consistently positive (e.i. after sing different M13 products, after all negative control in run) and/or you are otherwise sure the insert is right (sequencing, ...) one thing that pops in my mind is trying to use DMSO in reaction (1-10 %, though I usually get best results with 5 %) to check out whether this couldn't be the reason of secondary structures in the gene-specific primer binding site. This may IMO differ between complete bacmid and shorter PCR product, thus making the nested PCR positive.
But that's just a long hunch..

Sounds bit like an Impossible astronaut material..

[imladris]

Hi Trof,

Thanks for the quick reply. I really appreciated the Dr. Who reference.

As for my question/your suggestion: Sequencing indicates that the insert is present in the bacmid, in correct orientation (as suggested by the nested PCR) without any nucleotide changes. I will consider using DMSO. The one gene specific primer I tried could form a hairpin structure, although the other gene specific primer will not, so I am unsure why I consistently had the negative result.

[Trof]

Yes, the thing is, someone just day ago posted a paper about how secondary structures (and actually PCR efficiency) are affected by differential "nicking" of genomic DNA . If you imagine the same sequence in a long bacmid, it may similary have different secondary-structure characteristics than relatively short PCR product of the same sequence. That would be a pretty extraordinary probability, but I would be curious trying the DMSO, since it's easy to do, what if..