false positive in colony screening PCR? - (Dec/13/2007 )

Hi! I want to cut my insert out of one vector and ligate into another and even though it should be very simple it will not work.

When I screen my colonies for insert with PCR, all of them is allways positive, but when I miniprep them and linjerize them, the size corresponds to the vector.

So there is two problems: The vector does not have any insert...
i have...
1) run the opened vector on a gel and know that it is linjerized
2) remembered to dephosporylate the vector
3) tried some different vector:insert relations

anyway somehow it religates

And the other problem: My PCR gives false positives...
Decontamination is a likely cause but can it be due to the leftover insert-sequences in the ligationmix that been detected... in that case it is VERY diluted?? Is that possible?

So my main question is: Is there a risk to detect the templates for the ligation when screening colonies with PCR??
And if you have some other solutions for me you are very wellcome!

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Have you run a negative control with your PCR to verify that the so-called positives you're getting isn't contamination?
As for why it is religating - perhaps your enzyme is not cutting completely and you're transforming circular plasmid. It may be hard to detect if there is a small amount of uncut plasmid in your prep. Try running your vector out really far on a gel to separate linear from circular and gel isolate the linear plasmid.

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Some restriction enzymes tend to stick to the ends of the DNA fragments and prevent dephosphorylation. Try cleaning up the DNA before this step.

Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates. (Qing Dallas-Yang, Guogiang Jiang and Frances M. Sladek, 1998. Avoiding false positives in colony PCR. BioTechniques 24 (4) 580-582.) Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 e 10 molecules) of background insert are available for a 100 µL colony PCR assay. To avoid this problem, at least one primer should anneal to the vector. Use of the Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification.

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Do you have a list or anything of which enzyes? I used XhoI

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I used 0,135ug of my 7,2 kb insert --> 0,027pmol--> 0,00135 pmol/ul (20ul ligeringsreaktion)
Took 3 ul for transformation giving 1,35*10^-5 pmol/ ul (in 300ul bacteria+soc). I calculate with all of the insert unligated.
Took 25ul on a plate--> 3,38*10^-4 giving 6*10^-8 pmol/mm2 (using the same size as above). If one colony is about 1mm2 than one colony should contain about 35000 copies.

I used just a little of a colony, så in my 10ul tube there might be about 350 copies.
I know that real-time PCR can detect as little as 5-10 copies but I used gel-based PCR, 35 cycles. It can not be that sensitive or can it?

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As someone already said, colony PCR can easily give false positives depending on your primers. Are you using primers against just your insert? Even if your insert is incredibly dilute, PCR is sensitive enough to detect and amplify it. Using one primer to your vector and the other to your insert should eliminate the false positives, that way there can be no amplification unless the insert and vector are actually stuck together.

Regarding the re-ligation, how are you digesting your vector and insert? If you're doing 2 digests to open your vector/cut out your plasmid, test each digest alone to make sure they can effeciently linearize your plasmid.

In my experience, you can do all this and follow the rules perfectly, but if your DNA isn't clean enough or partially degraded the ligation is doomed. Cloning w/ clean DNA can be hard enough, trying to do it w/ degraded DNA will drive you insane. When purifying cloning inserts and vectors it a good idea to run your gels a little slower to make sure you get nice tight bands on your gel and detect any significant degradation. If you see something like that on your gel (smears or a really fuzzy band for your target), don't bother using it and try to get a cleaner product by trying the digest again, or prepping new DNA, etc.

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Thanks for answers!

Next time I will use a primer in the vector together with one in the insert. Sounds like a very good idea.

I had only one digestion, and my neg control (linjerized vector+ water) gave about the same number of colonies as for the one with the insert.

Anyway, I picked more colonies from my last try, used an other primerpair (not in the combination above) and all were positive again, but some were more positive, and they contained the insert. So now I have two colonies with the insert in the right direction tested by digestion cutting. (yes!)

But yes it drives you crazy when something you know should be simple doesn't work...
I tried my products on gel and they looked good.

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I realize that this is an old thread, but perhaps someone will answer my follow up question. First, let me say that this provides a good explanation for something I have known (empirically) for a long time. BUT it doesn't explain why true positives do not give a stronger band. We recently did a set of PCRs using primers only in the insert and got 24/24 weak (presumably false) positives as described above. After repeating the experiment with one primer from vector and another from insert, we got 10/24 strong (presumably true) positives. A couple of these were grown up and confirmed by digest. So, why didn't we see these clear, strong positives over the background bands from insert DNA on the plate??

Thanks!

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Why don't you think it's just the difference of PCR, I meant just by changing one primer, the yield of PCR product could be lower. Because in my cases, sometimes the insert primers amplified the stronger bands, compared with the vector primer-insert primer, and sometimes the reverse results appeared. As far as I think, this is the only reason.

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