I had extracted dna from E. Colli which shown very low digestion even with Hf digestive enzyme It is suspected that becouse of 1-3 minute kept plasmid with Pd3 during plasmid Isolation caused supercoilling of Plasmid which hindering restriction digestion
Heating does not remove supercoiling. It requires a nick or break in the DNA by an endonuclease or a topoisomerase or to remove it.
I agree with Adam B Shapiro that supercoiling will not be removed by heating. However supercoiled DNA will digest just fine with restriction enzymes. So if your plasmid is not being cut by your enzyme, the problem is something different. It may be that your restriction enzyme has gone bad (did you do a control reaction with some other DNA) or there is an inhibitor in your plasmid prep, or the RE site does not exist on your plasmid. You could try digesting with some other enzyme.
Hi there,
No, it requires at least a single strand break (nicked DNA). It can be done enzymatically with topoisomerase.
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