I am trying to distinguish between 393 and 374 band lengths by RFLP method. I am running in 3.5% agarose gel in 25x15cm gel electrophoresis to visualize the enzyme cut. I could not distinguish the bands with 60 minutes of running at 120 volts. When I ran for another 25 minutes, the bands faded, but the marker was visible. If I run it directly for 85 minutes, no bands including marker are visible in the gel. I also tried post staining method with ethidium bromide, but it didn't work. Any suggestions? Thank you...
Agarose gels do not have the resolving power to distinguish fragments that differ by 20bp.
You should run a 6% acrylamide/TBE gel which will be able to resolve the 20bp difference in your fragments. After running the gel, BRIEFLY place it in ethidium bromide solution and then destain in water. You may need to do several water changes during the destaining process. Be sure to include an appropriate molecular weight marker that will allow you to size the fragments.
You need at least a 10% difference in fragment length to resolve on an agarose gel.
It might be easier to genotype using Sanger sequencing instead of a digest & gel.
Good luck!
Begüm Beril Yalçın If your fragments have enough sequence difference, you can try Denaturing gradient gel electrophoresis (DGGE) or Temperature gradient gel electrophoresis (TGGE).
It's going to be a real challenge to separate your two bands. Standard agarose gels aren't going to be up to the task and if you don't have specialized equipment for acrylamide gels and so forth then you'll have to try something else. Any chance a different marker would be useful?
if you restriction digest the amplimer then you have a shorter product and better separation, for instance 100 and 120 would separate well on agarose
You can improve band separation in agarose gel if run electrophoresis in cold room (+4oC).
Ok, if the enzyme cuts one of your sequences but not the other, redesign the primers so that the cut/uncut site is in the center of the amplified fragment. Make it easy!
What are you trying to use the RFLP information to do? Identify progeny? Genotype individuals? Map a gene? If this specific marker ends up being a real time/reagent/effort sink then move on to a different one that fulfills the same general purpose.
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