How to get sharp band on agarose gel? - (May/31/2005 )
I need to purify my insert from agarose gel (0.8%), but the band of my insert (3.2 kb) is very close to the band of plasmid (3.5kb) because both bands on gel are not sharp enought.
is there any one who have any idea how to get very sharp bands?
is it a depeding on the voltage? running buffer, loadig buffer?
Try using thiner comb when making gel so the bands will be sharper.
Run the gel longer and at lower voltage.
That's my $0.02.
I would deff. run the gel longer - you will increase the resolution of the bands
You could also look for an restriction enzym that would cut just (once) in your vector generating smaller fragments of you vector.
Increase the agarose concentration, that gives better separation!
Another problem is overloading of the gel, this will lead inevitably to smearing
and overlapping bands. An additional cut in the vector is definitively the best
solution if your insert is not larger or smaller than approximately 20% of your
You should be able to separate these fragment sufficiently on a 1.5% agarose gel, run on a low voltage for as long as possible. If you set your gel in the fridge then this may increase the resolution of your bands.
Hi everyone, thanks for you all.
I have tried thin combs with slower voltage. it works pretty well