I did a double digest on a TA cloned vector and putted the insert in an other expression vector, pcDNA3.1+. As it is shown in figure, three first wells are plasmid extracted colonies for which PCR performed (476bp amplicons). Two following wells are non-template controls. What is wondering me is five additional wells. Those are wells co-responding Colony PCR of same colonies for which plasmid extraction and PCR is done (first three wells). But it is look like the pcr process are completely amplified certain vector. More surprisingly, in two right end wells of gel, plasmid extracts for which pcr performed and had desired band are smeared. I want to find out if my work is ok and insert is in the vector. I did plasmid extraction with alkaline buffers and phenol-chlorophorm-isoperopanol and ethanol precipitation.Sincerely
Hi there,
First of all, your PCR experiment is giving very low intensity bands (except the last 2 obviously but as it is overexposed I can't tell...) so there might be some issue there. There are also some controls lacking : colony PCR on a plasmidless clone and colony PCR on a clone containing the empty plasmid to see what kind of background is expected in the cell context. Intuitively I would say the last 2 reactions are true positives (if there are true bands with the expected size there...) and I think the rest you get is non target specific amplification.
Thank you dear Dominique. About controls I have done Colony PCR for them before; Colon containing pcDNA3.1+ whithot insert and no transfected DH5-alpha alone. PCR were negative for bout of them. In last step, TA coloning, I had the same, you called "non-target specific amplification" bands. About the size of bands, they are my actual desired size, 476bps. Ladder is 100bp but badly opened. Middle ladde's band is 500bps where my band is below it. I want to continue my work and transfet cell line, but I do not know take this results as possitive or no. What would you do?
Sincerely
Hi again,
As your colony PCR is not working great I would go for systematic plasmid extraction, digest and sequencing for those exhibiting the right profile of digestion.
I completely agree with Dominique. Use empty pcDNA3.1 vector as negative control and to check whether sites from the vector backbone gets amplified by your primers. Use the TA cloned plasmid as positive control to check the amplicon of your desired size. After you get positive result, digest your clones with the restriction enzymes you used for cloning, load them beside your colony PCR products and see whether they align according to your expected size. Moreover, if you have isolated plasmid and then set PCR, your bands (with insert) should be more intense. That is what I get after 29 cycles.
Hope it helps!
Dear Saha, I actually did digestion on TA coloned vector, after gel extraction I ligate gel-extracted digest in double digested pcDNA3.1+. My primers do have 5' overhangs for enzymes XhoI and EcoRI. I use Maxima Hotstart Taq DNA poymerase which in contrast to general Taq, has an annealing temprature 53 degree. General Taq had 65.5 annealing degrees. For both I performed annealing temperature gradient. DNA sequencing has shown exact amplicon coressponding desired noclutide sequence. For TA ligation I used Thermofisher kit. Everything went good till I saw this non-specific bands. So I think, as dear dominique says, we need to perform double digestion on the pcDNA3.1+ containing insert, which I din not do that yet. That was a very helpful consultation with you dear researchers.
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