I have done a digest with EcoR1that should only linearize my plasmid as it has a single cut site, but I am getting two bands & I do not think it is supercoiled plasmid. The top band is running at the correct size, so the bottom band is puzzling me. I did diagnostic digests that either cut once (HindIII), twice (Xho1), or three times (Nsi1). Strangely, the single cut with HindIII also yielded a second band, with the top band running at the correct size. (I am not getting the smaller bands with the two enzymes that cut multiple times but I believe that it just because I have very little product, as the large band is running at the correct size for both so I am not worried about that).
Any ideas as to why this could be? I have added an image of my gel.
Hello Blackburn,
I would like to suggest you, to run always a negative control for this kind of experiment in future i.e., (Plasmid + insert without digestion). Hopefully, there were no other pre-existing EcoR1 and HindIII sites in your plasmid and I expect your plasmid is not even contaminated! I hope you are pretty sure about the quality of your plasmid sample…
If these are all so, it might be happening because of two reasons:
1. If the plasmid has not been digested properly. It is often observed when you load a lot of DNA and compare to that if your enzyme is less for the reaction.
2. "Star activity" of enzymes - under non-optimal conditions, sometimes enzymes can exhibit nonspecific site cuttings. Hence, you should take care of the optimal conditions as well. I mean the optimal temp., buffer, time of restriction reaction etc.
Apart from these, I guess it is difficult to comment on; since; we do not have the negative control in the gel!
The below mentioned link may help you in troubleshooting further.
http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/34351/DNADigestion.pdf
Good Luck
Debasish
I think there is something odd going on here. If both HindIII and EcoRI are supposed to cut only one time, then the band should be the same size, which they are not in your gel. (I assume you mean the top band from the pair and not the much fainter band that is above your MW marker). One guess is that you have a DNA prep with a mixture of plasmids of somewhat different sizes. But as Debasish suggests, you might also just have partial digestion where the one band is linearized and the other is uncut supercoiled plasmid.
Star activity. if the topmost band is the correct size for all your cuttings, and you get smaller bands below, then it means the enzymes are cutting at non-specific sites. If you get bigger bands above your expected, then it means the digestion is incomplete. If you're using a lot of enzyme, try reducing it to maybe 0.5 units as high concentrations of enzyme (glycerol) may cause star activity. If that doesnt work, try reducing incubation time.
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