Hello,
I have performed some recombineering protocols and realised that the chances of my plasmid being in a multimeric state are quite high.
I previously designed 7 primer pairs that will produce alternating amplicons of 500 and 700 bp around my recombineered plasmid (which is 35kb) just so that I could get an idea that no weird recombination events occurred when looking at it in a gel.
Anyways, I did the 7 PCR reactions on a control with the original plasmid, and they produced the expected pattern, but when performing it on my miniprep-purified plasmid I was obtaining a lot of bands of all sorts of sizes (larger and shorter than expected amplicon). Funny thing is that these multiple bands seemed to follow the same pattern in all my replicates (different pattern for each primer of course, but same throughout the different colonies tested) which makes me rule out the possibility of salt contaminants affecting primer binding etc. I thought it might be bacterial genomic contamination that was being amplified, so I performed a CsCl-ethidium bromide density gradient to purify it and sent it off for sequencing.
But now Im wondering, would a multimeric plasmid yield multiple bands if amplified with a single pair of primers?
By the way, I can't run it on a gel to assess if it's multimeric because of its large size 35kb, although I am going to ask if anyone at my lab has a pulse field gel electrophoresis just in case.
Thanks!
Having multimeric plasmid DNA is probably not the explanation. While in theory you could have 1 primer binding at the one copy of your GOI and the reverse primer at the other end of your GOI on the other plasmid copy (in a dimer for example), in the case of your plasmid that would be very much too large to actually amplify. So I don't think that is the explanation.
You could digest your plasmid prep with a restriction enzyme that cuts outside the region you are amplifying. It doesn't really matter how often the enzyme cuts so long as your amplicon is not cut.
It might be that there are some rearrangements of your plasmid taking place if there is homology between the plasmid and the genome (and your strain is normal for recombination).
Seems to me you have some of the "weird recombination events" going on, that you were talking about yourself.
Did you think about analytic restriction digest to check for size/multimerization?
Yes, performing a PCR on a multimeric plasmid population can yield multiple bands. This is because multimeric plasmids are circular DNA molecules that can exist in various forms, including monomers, dimers, trimers, and higher-order multimers. When a PCR is performed on a population of multimeric plasmids, the primers can anneal to different regions of the plasmid and amplify different combinations of plasmid molecules, resulting in the production of multiple bands. The size and intensity of the bands can also vary depending on the distribution of plasmid multimers in the population and the efficiency of PCR amplification. Therefore, to obtain a clear and specific PCR product, it is recommended to use primers that are designed to specifically amplify the desired plasmid form or to perform plasmid isolation and purification to obtain a pure population of the desired plasmid multimer.
Hi all,
Thank you for your answers,
I did a restriction digest with enzymes that cut multiple times and indeed, this plasmid has recombined in all sorts of ways except the one I was planning on...
Thanks!
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