Isolated plasmid when mostly in supercoiled form eases most of the downstream processes, but is it essential for restriction digestion also? If there is less supercoiled and more of nicked circular or linear plasmid then it affects transfection efficiency, how does it affect digestion efficiency? Will it be not digested at all? If digested, can't it be used for cloning purposes?
Hi there,
Restriction enzymes are the most effective on the less structured DNA. And contrary to the upper message, there are cases where supercoiled DNA is far less susceptible to digest because DNA structure may hide restriction enzyme cleavage site. NEB has established a table comparing enzyme activity on supercoiled plasmids and on linear lambda DNA:
https://www.neb.com/tools-and-resources/selection-charts/cleavage-of-supercoiled-dna
Digestion with restriction enzymes is not affected if the DNA is supercoiled. For greater efficiency heat the DNA at 94 ° C for 10 minutes to break the supercoiled structure
Hi there,
Restriction enzymes are the most effective on the less structured DNA. And contrary to the upper message, there are cases where supercoiled DNA is far less susceptible to digest because DNA structure may hide restriction enzyme cleavage site. NEB has established a table comparing enzyme activity on supercoiled plasmids and on linear lambda DNA:
https://www.neb.com/tools-and-resources/selection-charts/cleavage-of-supercoiled-dna
Even if you suspect the efficiency of digestion by enzyme, u can linearize DNA and then digest.
Hi Saha,
True. I agree with the above answers. I would say that during restriction digestion, the concentration of DNA matters more than the form of DNA template taken. Depending on the type of DNA template, the concentration needs to be adjusted to get efficient digestion products. Even if you find some undigested vector on the top in the gel, (In the case of supercoiled DNA as a template), the required digested product (band of your interest) can be gel purified and can very well be used for cloning purposes, It works....
Hi Tias, Dominique is correct regarding restriction digests.
Regarding transfections, supercoiled DNA transforms ~100x more efficiently than linear (relaxed/nicked is intermediate). This is due to the increased space that the relaxed and linear forms take, making it more difficult to a charged molecule through the lipid membrane. However, linear DNA integrates ~100x more efficiently to give stable transformants.
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