Colony PCR positive, miniprep negative? - (Mar/10/2006 )

Hello everyone,

I wonder if someone would help with the following:

Ive trying to clone a PCR fragment of ~600bp into pSTBLUE-1 from Novagen. After liagtion and plating, i do colonie PCR to check for positive insertion (i dont do blue/white screening). This is the strange bit. The one´s which are positive on PCR, i grow them in LB overnight with carbenicillin (50micro/ml) and SpinPrep them. The EcoRI digest should popout the insert, BUT unfortunatly so far I only get negative results!! Would anyone have an idea on this??

Thanks
Filipe

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Try picking your transformants from the transformation plate to a grid on a fresh plate. Grow them overnight, and screen those colonies by PCR.

We routinely do this now, because we were getting an unacceptably high number of false postitives when screening transformants directly from the plates on which the transformation was plated. It made a huge difference.

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But How can a colonie be PCR positive (thus having an insert), AND after o/n growth and plasmid prep THE SAME colonie has no insert (based on EcoR1 digest)?

Thanks
Filipe

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There's the logic flaw. The colony never had an insert -- the colony PCR was a false positive. The PCR product arises from something other than the fact of a correct clone. We see this all the time if we do colony PCRs directly from colonies that arise on the transformation plates. I'm not exactly sure why this occurrs -- perhaps insert DNA is carried over from the ligation reaction used to transform the cells.

While I'm not exactly sure why this occurs, I do know we solved it by first patching the colonies to a fresh plate and then doing the colony PCR on these.

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Thanks for the suggestion. I will try it.

Filipe

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i would suggest also try digesting with different restriction enzymes....sometimes using same enzymes that you had desinged by PCR doesnt work....do you have any flanking sites?

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Kathy ahs a good point -- were the restriction enzyme sites that you're using to clone your fragment engineered into the primers? Do you know that they're correct? Does the plasmid isolated from your PCR-positive colonies appear larger than that of vector alone?

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Hi Filipe,

Yes , often this is a problem .So better not to rely on COlony PCR. Please do RE check and sequence check to find out your insert.

Saikat Chakarborty
Mie University
Japan

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How are the primers positioned for your pcr? One should be on the vector sequence and one the insert.

I suppose if you used primers that were both found on the insert, you could have insert DNA from the ligation carried over onto the plate. Then when selecting colonies you pick up the insert as well and get a false positive.

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Thanks everyone. Ive now sorted the problem. Simply by just buying the PGEMTeasy. Worked first time same conditions!!

Thanks again
Filipe

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