Hi. I successfully cloned an insert into a plasmid vector and subsequently transformed it into E. coli. However, upon sequencing, I discovered that the insert-plasmid sequence is repeated two times, making insert-plasmid concatemer.
Do you have any opinion about this issue?
Thank you in advance
Dimers just happen sometimes during cloning. I would try sequencing another few positive clones. If they're all dimeric concatemers though then something might be wrong in the plasmid backbone you're cloning into.
So, to clarify, you ended up with a double sized plasmid? Two backbones and two inserts? Or one backbone with two inserts?
You could try a few things. Lower the concentration of the insert, or ligate the same amounts and ratios of DNA in a larger volume.
Depending on the sizes of your backbone and insert you could try a quick plasmid prep and DNA gel to check the size before sequencing.
Amy Klocko Thank you for your advice. Yes, apparently, there is a recombinant plasmid with two backbones with two inserts.
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