I've been struggling with plasmid digestion. I've tried different plasmids, pUC19, pBBR-MCS2, pKD46, with and without cloned genes. For now, I want to single digest them, just to linearise them and make sure of their sizes, so I've been using EcoRI or BamHI. I use the appropriate buffer and enzyme concentrations, for 500 ng of DNA, 37 celsius degrees, 30 minutes, and when I try to visualize the digestion in agarose gel 1%, there's no bands. However, I always run the plasmid digestion gels with a control, that is, the very same plasmid, but before digestion. Then guess what? The not digested plasmid looks beautiful.
I've changed the water, wondering if there was any DNAse in it. I've tried to digest PCR amplicons as a control for plasmid digestion and the digestion kit just looks fine and I got the desired bands.
The thing is, if my digestion is not working, shouldn't I, at least, find bands the same as the not digested plasmid? If the plasmid extraction is leading to denatured DNA, shouldn't I be seeing many small length bands? Could it be any interaction between the digested and the loading dye buffer? I've already tried to purify the digested plasmid before visualising it in gel. No bands.
If my digestion is not working, shouldn't I, at least, find bands the same as the not digested plasmid?
Yes.
If the plasmid extraction is leading to denatured DNA, shouldn't I be seeing many small length bands?
Yes, you should see 'smear'.
Could it be any interaction between the digested and the loading dye buffer?
Yes, if the loading dye contaminates with substance which can degrade DNA, then your DNA can be degraded.
Are the restriction enzymes or restriction enzyme buffer(s) contaminated with DNAse? Borrow some restriction enzymes /buffers from other lab, and do it one more time. And then update us.
Hi Manuella,
For troubleshooting you can try on changing the buffers and enzymes to check for DNAses contamination as the answers above suggested.
However, as you didn't offer any details on your electrophoresis conditions I would risk on a different hypothesis. As the undigested plasmid is a supercoiled DNA it will run faster in the gel that your open-circular plasmid (as it will be if digestion was suscessful) so you will "see" your undigested plasmid sooner in the gel. You have to take that difference into account when running the gel and analyzing the results. So, for more troubleshooting, you can try on different electrophoretic conditions (like the running time, or the agarose percentage) to be sure you have covered all possibilities.
Good luck!
Annia
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