Hello everyone,
I'm digesting 2 ug of midiprep isolated pET15b and 22b with BamHI and XhoI at 37 oC for overnight. There is smearing in gel and size of the cut vectors also not appearing actual, around 5.4 kb for 22b and 5.7 kb for 15b. I used 1 kb Trackit DNA ladder from Invitrogen and concentration of agarose gel was 1% in 1X TAE buffer.
I think the main problem you have is that the gel is overloaded, the samples are in excess. The smear is most likely from some genomic DNA contamination in your plasmid prep. But if you loaded less plasmid then the smear would be greatly diminished. I would say that both the ladder and the samples are about 5-fold in excess.
It is hard to tell whether in addition to overloaded samples, there is a also a problem with the gel itself. It appears a bit anomalous, but that might be due just to overloading. So try running it again with much less sample and see how it looks. Then you can begin to analyze if you have a real problem or not with your plasmids.
I do agree though though that one of the plasmids looks wrong in terms of size.
What buffer did you use for your digest? Cutsmart can exhibit star activity with BamHI so I'd suggest doing 3.1 if possible. Otherwise I don't know what your problem could be. I agree with Michael J. Benedik as you need to significantly lower the amount of DNA you loaded.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning