Hi!

I have 2 plates of a single species that I’m preparing for ddRAD. Both were extracted using the same kits around the same time and normalized to the same concentrations (20 ng/ul). One of the plates proceeded fine through the procedure, but the 2nd plate has double bands popping up after the PCR (attached). The methods I’m using are:

Digestion & Ligation in the same step, 12 unique adapters

Bead cleanup

Qubit

Pool within an index group

PCR (10 ul DNA per rxn), 8 index primers

Gel

Bead cleanup

Size selection

Would anyone have any idea why I'm getting double bands in the gel after the PCR only with the 2nd plate? I have since gone back and re-done an index group from the 1st plate using the same reagents and had good success. My best guesses are that 1) ligation is not going well, as I've also run a gel using pre-PCR bead-cleaned product and it showed a nice smear, or 2) something is wrong with my raw DNA.

Thanks for any insight!