Hello,
For one of my project, I have to clone genomic cDNA fragments (8kb) of SARS-Cov2 virus in a plasmid (5kb, high copy). I amplified 2 cDNA fragments (2 x 4000 pb) through PCR and I used In Fusion cloning kit (Takara Bio) to insert it in my vector (see picture). After ligation reaction, I transformed Stellar bacteria (classical heat-shock protocol) and spread bacteria on LB + Ampicillin agarose plate. To test if my colonies have integrated my plasmid + inserts, I performed a colony PCR with primers targeting genomic SARS-CoV2 fragments (product PCR = 1500 bp). I've got several PCR positive bacterial clones, thus I put them in a LB + Ampi culture medium and extracted the plasmids through MiniPrep kit (Sigma). I sequenced it and all my "positive clones" carry actually an empty plasmid.
1- I tested my PCR colony protocol on bacteria transformed with empty plasmid --> Negative results
2- To decrease empty plasmid contamination, I dephosphorylated my linear plasmid and tried again ligation/transformation protocols --> PCR positive clones obtained but again empty plasmid after MiniPrep extraction.
So it seems that my positive clones lost their inserts after only a single passage in LB medium. I suppose that the combination of plasmid size (13kb) associated with a high copy ORI is potentially letal for bacteria. Thus, they cure large plasmid to keep the "contaminating" empty plasmid.
What do you think about it? Do you have a solution to quickly fix this issue ? (another bacteria strains for example).
Thank for your help.
I believe the reason for high background with recombinantly negative colonies is because the ligation has one of the component with a large molar excess, usually the smaller fragments. I'd repeat everything but decrease ratio of smaller fragments but grow bacteria at 28 degrees Celsious. Don't screen with PCR, isolate plasmids and they should be good (stable plasmid with right orientation).
do your pcr primers to check uptake go from vector into insert or sre they just insert primers?
If just insert is it possible that you have spread template dna on the selection plates with the cells and your positives are just wetting effect on the cells so initially pcr positive but when grown up and diluted the colonies are negative because they always were
I am inclined to agree with Oscar, particularly with respect to using PCR as a means of screening for inserts. In addition to the problem that Paul mentioned - that there may be residual Covid DNA on your plates, any recombinant plasmids colonies you do produce are potent contamination sources in themselves. High-copy plasmids don't fit in well in a PCR laboratory.
Thank you very much for your answers, I will take that into account and change some parameters in my strategy.
Best regards,
Clément
Well, I performed a new test where I reproduced again the same ligation/transformation protocol (with another bacterial strain) but I added in my colony PCR a scrape of my agar plate without bacteria as negative control. The PCR was positive ... Thus, Paul was right, I definitely have a DNA template contamination. I stop colony PCR and will follow Oscar advises' for the ligation protocol.
Again, thanks a lot !!
An inconvenient problem, but it's handy to know it has been experimentally confirmed. Thanks for sharing.
Andrew
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