I cut 500 ng plasmid in volume 150 ul with 15 ul enzyme Hind III fermentas (exp 2010).
Incubate 37oC, 3h after denaturing 37 -> -20 -> 37 -> -20 oC to increase number of linear plasmid
-> incompletely cut. It still had linear and supercoil forms
-=> add more 10 ul enzyme, incubate 1h -> Supercoil form was still here. :(
I know the enzyme was expired, but I dont think it cant cut efficiently with high concentration concentration like that.
So, may you suggest me some solution for this problem, and some way to denature plasmid?
Thanks in advance
Hi there,
Have a go with a new batch of enzyme and try to respect manufacturer's instruction in terms of DNA and enzyme concentrations. There is no need to force the dose of enzyme : the only thing you could get out of it is non specific cutting. To get sure your batch has run off, digest your plasmid with another restriction enzyme for which you know there is an unique site in your plasmid and which has not expired.
Hi there,
Have a go with a new batch of enzyme and try to respect manufacturer's instruction in terms of DNA and enzyme concentrations. There is no need to force the dose of enzyme : the only thing you could get out of it is non specific cutting. To get sure your batch has run off, digest your plasmid with another restriction enzyme for which you know there is an unique site in your plasmid and which has not expired.
"It still had linear and supercoiled forms"
If it is linear, it has been digested. Maybe you mean "relaxed"? Maybe not.
If it is cut, then the enzyme is still active, and you don't need to denature the DNA, which most people would understand as converting to singlestranded, which of course will not be digested by the RE.
Of the possible explanations, I suggest an impure (mixed colony) plasmid.
If you are interested in obtaining your plasmid in a relaxed, circular form, you can treat it with a topoisomerase enzyme.
Of course, once it is cut by a restriction enzyme, it will be linear, and therefore relaxed. If you religate the cut plasmid with ligase, it will be relaxed and circular.
I imagine you want to obtain the linear form. If it is so easy for you to differentiate this one with the supercoiled one, I guess you are running a gel. Can't you just cut the linear form from the gel and work with it?
We usually digest 10ug plasmid DNA in 100ul volume using 5ul of HindIII (10IU/ul)) for 10-12h at 37C. Never had any problem with incomplete digestion. So, my sincere suggestion to you is leave the reaction for longer period (overnight) at 37C rather than adding too much enzyme. There is no need to denature (or convert?) supercoiled plasmid for HindIII digestion. Also, make sure HindIII site present and intact in your plasmid.
Treat with Topoisomerase enzyme and then carry out restriction digestion
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