I have some questions for gel electrophoresis. I am now determining bp of sample
ID: 162640 • Letter: I
Question
I have some questions for gel electrophoresis. I am now determining bp of samples band and making a restriction map.
I used pUC 19 (2686bp) for this experiment, and cut it by two enzymes; Ava2 and Pvu2. Theoretically, two enzymes cut the plasmid to 4 fragments: 1209,933,322, 222 (bp). However, after running gel, i got totally five bands: 4 clear bands and very thin band at the bottom of the gel. I can't understand the fact. I tried to think that incomplete digestion of enzymes, but even if it happens, the number of bands only decreases not increases.
In additon, the sample's total number of bp is quite higher than 2686 bp.
how can I explain this prblem?
Explanation / Answer
There are two possibility for that extra band
1. Partial or incomplete digestion sometime vector is too supercoiled to open up and get digested. you can run a little undigested plasmid next to your digested one and if you see the same band there this will explain everything.
2. Star Activity may be also a reason for that as you mentioned the name of enzymes they have star activity so they can also digest nonspecifically in unfavouable condition like glycerool content and etc.