I am using an un-optimized touchdown PCR to screen a couple of microsatellite primers designed by a commerical company. The results aren't looking good and are not consistent at all.
I got this smear which I thought could come from loading the negative after the last sample. Negative is the one which is in between 2 empty lanes.
So I added the negative first. And it seemed alright except for the primer dimer?
So I assume all's well and tried it again without changing anything. Negative in the first lane followed by ladder.
IS THIS A CONTAMINATION?! Looks awfully like one. I had one previously and got new reagents, took the pippettes apart, clean them and recalibrate. WHAT IS THE PROBLEM?