Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Why do I get two bands on the gel of a digested PCR product after purification w - (Apr/27/2018 )

Hello friends.
I am trying to clone a region of 80 amino acids with a weight of 265 bp in pET28a.
When I perform the PCR of my insertion, I see a single band.
Perform the digestion of the insert with the NheI and BamHI enzymes and, when you check the result in agarose electrophoresis, show a single band corresponding to 265 bp.
Then purify the digested insert of the agarose gel using a kit. Take 5 ul of that purification and look at it on an agarose gel to verify the purification. Now two very close bands appear instead of just one.
What do you think may be happening?
Is it advisable to use this product to link it to the vector?
Thank you in advance for your help.

-Delimar Rodriguez-

I realize this answer is too late to help, but for any other readers, I would have to say that gel pictures would be of use in diagnosing the problem. It is possible you had two close bands (a "doublet") all along, but you could not see it because the mass of DNA on the gel was so great it made the two bands appear as one. When you only ran a little of the purified product, it was dilute enough to show as two separate bands.  The digest could have been incomplete, leaving one band a bit longer due to still having the undigested termini intact.  I assume you are using a high % agarose or using a special agarose for separating small fragments.  But I'm guessing- pictures are worth a thousand words!