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restricted PCR plasmid runs slower

cloning restriction agarose gel

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#1 Wickerl3732

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Posted 12 February 2013 - 05:59 PM

Hi everyone.

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.

#2 bob1

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Posted 12 February 2013 - 08:16 PM

Yeah, the REs can stick to the enzyme, which would retard the progress. Try heat denaturing the reaction before loading it on the gel.

#3 Wickerl3732

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Posted 13 February 2013 - 04:09 AM

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.





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