Bright bands of PCR products stick in gel wells - (Oct/15/2011 )
I performed PCR to confirm my gene knock-in is working fine. The templates are yeast genomic DNA. Primers' Tm are 45C and 60C, and I set annealing temperature at 55C for 30 second. The target PCR products should have 2400 bp. The genomic DNA has been 100-fold diluted before PCR, which is about 1-5 ng per 20ul reaction.
I have used new 1x TAE running buffer and another PCR product which the size is known (3300bp) as control.
But when I run a gel, all my PCR products just stick in wells and they almost didn't migrate. My control PCR product (3300bp) migrated at the right size. Marker looks normal.
Please give my any suggestion.
Do I need to dilute more for the template genomic DNA?
Thank you for the help.
I'm pretty certain you are seeing genomic DNA. You can test this by running a lane of your pcr reaction prior to cycling. I would dilute the DNA another 10-100x.
Your real problem is likely the 45C Tm primer, which probably won't work, or will be marginal if it does. You could try lowering annealing to 50C, but that still might not be low enough, and PCR reactions don't work, in my experience, with annealing at lower temperatures. I'd strongly recommend redesigning that primer.