TBE/Agarose Gel Concentrations - (Feb/18/2011 )
Hello everyone. I was wondering if any of you could offer some help on a cloning project I have going on. Basically, I'm trying to gel isolate a 3960 fragment from the gel. However, due to incomplete digestion I always end up with my linearized plasmid which is 4401 bp long. That's a difference of 441 base pairs. Last time I tried this project, I had to have help from the PhD student in our lab. I was wondering if anybody knew of any resources I could use to find out how the agarose % of a gel affects migration of the bands. For example, would a 2% gel allow for a larger distance between the two bands compared to a 1% gel (or vice versa)? Thank you for your input!
As our lab always says, "May the clone be with you."
Typically the larger the fragment, the smaller % gel you should run. For a 4kb fragment, typically .7-1 % agarose gel is the size to run. You may be able to see greater separation/resolution going with a higher percent, but its going to take a lot longer to run and may not even migrate through the gel. I would suggest going with a 1.2% or 1.5% at max and staining the gel after the run to ensure that the stain doesn't run past your DNA (E-Br runs opposite the DNA).
Try Roche Lab Faqs, especially the "Working with DNA chapter".
Thank you for your help! I'm going to try a 0.8% gel today and see how that goes.