electrophoresis resolution - separate DNA of similar size - (Dec/06/2001 )
I've got 4 DNA fragments to separate. the bands are 3040, 2953, 2763 and 2658 bp in the same PCR tube. I've tried an 0.5 % agarose in TAE with low voltage (50 volt) without any results.
I need your help. thanks
You may have to switch PAGE. I've only used these gels a few times and don't know very much about them, but I do know that they give better resolution than agarose. As an example, think of a manual sequencing gel, it is polyacrylimide and it can give resolution down to single bases. I don't know if the gels can handle such big fragments, though.
you need high percent gel, try 2-3%.
Polyacrylamide gel is good for such separation. However due to such large DNA fragments you are dealing with, you may need a lower % polyacrylamide gel, maybe 2-3%. I use 6% PAGE for 100 - 300bp.
If you want to use agarose, you may need a LONGER gel length with lower % in order to resolve the fragments.
Like the others here, I'm not sure that you fragments are too big to fit on a polyacrylamide gel... I use those gel for fragments under 500 bp usually.
I think that a 2% agarose gel run at a low voltage is all that you need with the fragments you have. 0,5% is really not enough, it is more suitable for 10 kb fragments... and do not load too much DNA on the gel as the bands will get bigger and less define.
I was thinking on my answer... 2 % agarose will be too much, the fragments may have problem to migrate at their lenght. I would try 1,2 -1,5% agarose. What is clear is that by increasing the % of agarose, you'll get a better separation until you reach the limit at wich the fragments won't migrate well because the gel is too tight.
Another thing to try is TBE instead of TAE as buffer. Some people think that TBE gives a better resolution. Personnaly, I don't see a lot of difference, but if you want to try...
Use 2--2.5% agarose gel, run with TBE buffer at very low voltage,around 50 v for long time.definitely it separates.
use less amount of DNA for loading.
try it,good luck
I agree with most. Try 1.8-2.5% agarose in TBE buffer. I have used this for ampliying simple sequence repeats and get good separation down to 1000bp. In addition make sure you use quite thin gels and run for a good length of time