Hi everyone,
I am trying to clone a CRISPR sequencing library. After transformation by electroporation, I picked single colonies and did minipreps, then digested with a single cutter enzyme. Most of my colonies look good, with a single band at the right size. However, some of my colonies have the correct band, plus three additional larger bands. What could be happening here? Could it be concatemers or incomplete digestion? Could my single colonies have two plasmids, one at the right size and one much bigger that's not being cut?
Any advice on what might be happening, and how to figure out what's going on with my plasmids, would be really appreciated. I want to know what is in the plasmid ideally!
Thank you
Hi there,
I would go for uncomplete digest... Check with either extending digest time or adding more enzyme. The other possibility is to go for a double digest to separate insert from plasmid backbone.
If you really, really want to know, then sequence the plasmid.
But since you have several colonies that are exactly what you expect, just choose those ones and continue with the experiment.
Good luck!
Try running cut and uncut plasmids side by side. If the larger band is from partial digestion, you will see that band size in all uncut samples. If it's still a puzzle, don't use those odd ones for analysis.
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