restricted PCR plasmid runs slower - (Feb/12/2013 )
I am preparing a PCR product insertion into my vector. It is 1080 bp product, and I am restricting at the 3' with HindIII to cut off 76 bp and on the 5' end I introduced a EcoRI restriction site with my forward primers, allowing me to cut off 11 bp.
But here is my problem: after restriction and analysis on an agarose gel I had the opposite which I would have expected. The 1080 bp PCR product runs at the right size, but after restriction with HindIII the band appears to be a little bit higher and after double digest with EcoRI, this band was even higher.
Do you guys have any reasonable explanation for this. I was guessing that the restriction enzyme might stick to the DNA, or that a conformational change may cause the higher band (?).
Paralleling the restriction of the PCR product I digested the plasmid with the very same restriction enzymes, and this digest was perfectly fine.
Yeah, the REs can stick to the enzyme, which would retard the progress. Try heat denaturing the reaction before loading it on the gel.
Thanks for your answer. I did the heat denaturation step before loading the gel. I think for some enzymes they use SDS for release, I will try that.