Easiest fix is to cut and gel-purify the band that you want. Minimize the size of the agarose around the band, make sure it completely dissolves in the first buffer, and don't overload the column.

Good luck!

Hi there,

I would go for the in vivo experiment and consider the potential negative effect of the contaminant only if it fails...

Hi Ana,

First, we can remove nonspecific bands of larger size by tuning the PCR conditions (like reducing elongation time, increasing annealing temperature, less cycle of PCR run, low template DNA concentration or using DMSO). These tunings will work I guess.

Secondly, you can go for re-PCR with your purified product.

Finally, consider the minimum gel portion during cutting. Try to avoid any contamination. I hope these will work, as your nonspecific band is too far from the desired one. So, there's a little risk about it.

Since your odd band looks about twice the size of your expected I wonder if the slightly denaturing nature of dna binding to the purification column is allowing 2 molecules to bind together at some common sequence within the amplimer giving a small amount of product dimer. You could try a simple purification like freeze and squeeze on the excised correct band and then see if the larger band can still exist