I'm doing RFLP analysis using enzymes RsaI and MnlI (from NEB) for polymorphism in IL-10, but the restrictions results don't show in gel electrophoresis, I've done as the protocol said,
5-15 minutes in 37°C for the inactivation 20 mnt 65°C, and other treatment based a paper in the same analysis from ju won roh, but it's still not visible, can anyone give me some advices?
Dear Atina, maybe you can increase incubation time and the amount of DNA or PCR product or restriction nz. We usually concentrate the digested DNA by NaAcetate mehod, then we run electrophoresis for RFLP comparison.
Best regards.
Dear Mert,
Thank you for your advice, we'll try to concentrate the digested DNA by NaAcetate method since we've increased the incubation time up to 2 hours bu still have ho results,
Do you mean you
A) can see DNA but it is not digested or
B) you can't see DNA at all?
If A) you may need to clean up your PCR product (assuming you're using amplified DNA as your starting material) as some restriction enzymes are fairly sensitive to buffer composition.
If B) then simply use more DNA, just leave the restriction digest on for a little longer (rather than put more enzyme in) and ensure you load all the reaction on the gel. For a given molar concentration larger DNA fragments will show up better than smaller fragments and as IL-10 is fairly small your fragments will be even smaller so it's probably worth running a 2-2.5% agarose gel which will concentrate the fragments into tighter bands to make them easier to see.
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