Hello everyone,
I've been trying to clone a bacterial protein from S. aureus into E. coli with pQE30 vector.
After ligation and transformation (with Xl1Blue), I screened my colonies (more than 30) and ended up with just two positive clones. However, one of these clones seems to have both the empty vector and the ligated product (see picture).
Is it possible that this clone during transformatiom did uptake both a re-ligated vector and the vector+insert? I'm pretty sure that I did not pick up two colonies instead of just one.
If they have both of them, can I continue with sequencing to check if my insert is not mutated? If I send to sequence a mixed sample with both the empty vector and vector+insert will it work? And if my insert turns out to be okay and I go on with transformation of BL21 cells and purification will I be able to obtain my protein even if I have an empty vector?
Anyway, I'll try to do another ligation and transformation to obtain more positive clones hopefully.
Thank you in advance!
This double band pretty much is the result of contamination by amplification product from previous runs. The same primers used for screening of various inserts in the same vector can be easily contaminated by amplicons from other experiments. However, other reagents can be contaminated too. Usually, it happens with amplicons from empty plasmids because they are relatively short and produced from the most colonies. When these short products happen to be in a reaction mix with suitable primers, they will become a good template concurring with the actual plasmid from a picked colony and producing double bands. It is safe to purify and sequence plasmids corresponding to these double bands because they are actually not a mix of two different vectors.
It could be cross contamination or other contamination or it could be that plasmid prep is mixed. I would suggest to take that plasmid that shows the positive band and retransform (using very small amount of DNA) then screen those again. Hopefully you will get some colonies that are now pure.
Always be sure to have a negative control (no template added) to be sure you aren't getting some contamination with vector.
In addition to the previous responses, if you rule out primer contamination I also suggest trying to re-isolate the positive colony. Sometimes we think we got only one colony, but what looks like a single colony is a merge of two very close colonies or two overlapping colonies. You can try re-streaking your positive clones and screening colonies from that. This will help explain what's going on.
You may also have both the empty vector and the plasmid of interest in the same bacteria (bad luck!). If that is the case you should probably start over the transformation. If you move on with it, your cells may end up spitting the plasmid and keeping the empty vector for antibiotic resistance (since the empty vector is small and less energetically costly for the bacteria to maintain).
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