Agarose Gel Electrophoresis - Troubleshoot (Jan/18/2007 )
I am having some odd problems with the results of my gels. I loaded 10 restriction digested DNAs into 10 wells and after the run (110V, 45min; 90V, 60min), I got a smiling band resulting from the first and the last bands being slightly distorted, like the following:
( _ _ _ _ _ _ _ _ )
Second, the DNA bands, as well as those of the markers, are lighter than the upper ones.
Have you guys had any of such problems? Please help.
not sure what you mean from the first question... never use the first and last wells in a gel, they'll turn out funny (some of the time).
Second, the DNA bands, as well as those of the markers, are lighter than the upper ones.
that's the way of the world. the bands of smaller fragments are always lighter then the bands of larger size fragments.
V
Are you using 10 different REs? Some of the RE buffers may affect DNA migration in agarose gel. May be you can try to purify the digests before loading..
Have a nice day..
VETTICUS, the lower bands are not as faint as with my previous gel runs.
WHY, I'm using the same RE. I ruled out the possibility that the RE and/or its buffer is the cause because I did not have this problem in previous gel runs and with my other colleagues.
Thanks anyway.
The problem is that you are running at to high V, is better to run between 80-90V (big gel, for a tiny one no more than 75V). Is better to run a gel between 1h45min-2.5h patience is the key for a good gel. I learn that after many gels that end in the trash, if you don't have time, then don't run it.
If you run your gels with ethidium bromide in the gel, it has a positive charge and will run towards the cathode (-ve) at the top of the gel. This means that bands at the bottom of the gel often appear less bright because there is less ethidium bromide there after the gel has been run. Lower bands can be better visualised by soaking in 10-20 ug/uL ethidium bromide after the gel has been run.
I do agree with you. But sometimes it doesnt work that way. I ran 100V before using small gel and it turned out to be very nice. Sometimes even at 75V, I still got the smiling effect. Anyone can explain that?
So I am not really sure whether voltage makes a difference or not.
the difference could be caused by the medium that your sample is in, ie salt, buffer, cofactors, etc.
i tottally agree with merlav.. lower voltage longer time is the key.
you are pushing ur dna too much
Oh.. it makes sense now. The quality of the TAE buffer. THanks
