OK, I have a good one. I consistently amplify a 1kb region spanning the ITS1, 5.8S, ITS2 using proven primers (primers 1 & 4). The PCR is almost bullet proof...until a few weeks ago. I started to get long smears with the sample appearing to be still caught up in the well. I've tried everything, brand new dntp, water, taq, even ordered new primers. I've used different pcr machines, diff. concentrations, etc. I got frustrated and decided to use a different set of internal primers that will amplify the same target in two fragments (primers 1 & 3 amplify the fist half, and primers 2 & 4 amplify the second). I didn't really think it would work because the non-working primers are still being used with a new partner (ie same forward primer, new reverse and vice versa). Low and behold they both amplified first try, with tight bright bands. So I tried again with primers 1 & 4, using the old set and the new set i ordered after problems started. Same smear appears for both reactions. So i know it is a priming problem, but why did these primers work perfectly and then stop working altogether? More confusing, why do they work separately?
I noticed another post on this site that had the EXACT same problem but nobody really answered the thread. He may have explained it better:http://www.protocol-...osts/23479.html
Also, it is definitely not too much template which is causing the problem.
Any thoughts would be great.