eternalmetal

I had an impurity in my sample after a BglII digestion followed by CIP. To attempt to assess the problem with a quick gel, I ran in the first lane the original plasmid straight from the miniprep, a sample containing the plasmid cut by itself, and in the last lane the sample cut with BglII and CIPed, showing the impurity at around 2kb. the marker is a 1kb ladder with bands at 10, 8, 6, 5, 4, 3, 2, 1.5, and 1k and 500 bp. Here is a picture of the gel im talking about: Now I know that when you run an uncut plasmid you typically get 3 bands. Nicked/open, linear, and supercoiled. The sample however contains those 3 bands, and a 4th unknown band. Note that when I cut with BglII, I am only supposed to be cutting at one restriction site, so there should only be one band. But when I cut this plasmid I get an unknown 2 kb band. My guess is that I have an impurity somewhere from the miniprep, but what could it be? Im using an E.Z.N.A. plasmid miniprep kit to prepare the plasmid. Any insight is appreciated.

What would you clearly see if the plasmid was underdigested? Im assuming if it was digested too much it would look like this: I didnt see any extra bands, but I guess if I wanted to verify if it was digested I would have just run an undigested sample side by side for comparison. There werent any problems with the experiment (as far as I know so far), this topic was purely speculative so that I could understand more about enzyme digestion. Thanks again for all the help

I used the correct buffer without diluted enzyme, and yea I meant 2 ug The rationale between using more enzyme was that an experiment someone else did 6 months ago did not turn out, and who I report to thinks that the underdigestion of the plasmid was the culprit. The goal is to open up the plasmid in order to add an insert and transform into e. coli. Im no expert, but I dont think that this type of application would require extra digestion as compared to your typical experiment. Or does it? I just dont want to learn by making a mistake that I was led into making. If it were up to me I would have used 2 uL of enzyme. Thanks for the reply Charon

I am running a restriction digest with the Pyp04 plasmid (has 1 restriction site for BglII) and BglII. Using 2ng of plasmid, I was told to use 5 uL of BglII to digest. After 2 hours of digestion I ran a gel to test how well my sample was digested, but did not include an undigested sample (I am under the assumption you wont just see one band if it was under digested). The bands were a little on the light side, so I am doing an etbr soak to brighten the gel up. In the meantime (1.5 hours after I started the gel), I was told to add another uL of enzyme to the digestion for another hour. So my question is, would it really be necessary to do so? A typical digestion of this amount would be done with less enzyme and would most likely be fully digested in an hour's time. Im kind of new at this so im just looking for a "what would you do" type of answer. Imo adding an extra uL of enzyme would be unnecessary based on protocols so I didnt add it. Opinions? thanks in advance