3 replies to this topic

[LabLackey]

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."

[Zach T]

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).

[bob1]

Try Roche Lab Faqs, especially the "Working with DNA chapter".

[LabLackey]

Thank you for your help! I'm going to try a 0.8% gel today and see how that goes.