I'm amplifying sequences 200-250 bp in length with the intent of sending them to sequencing for determining if an SNP is homo/heterozygous, so I need fairly high quality single length product. I have three different problems:
1) With several samples, I got multiple bands in my gDNA, but not in my cDNA. Okay. I've tried re-running the PCR at a higher annealing (from 53-56 degrees Celsius) to decrease non-specific binding, but merely got lower volumes of the multiple bands or nothing at all (the primer probably degraded). Ideas?
2) For some of the samples, I cannot get any product at all for the gDNA, but plenty in the cDNA. I thought maybe the primers were degrading, so I decreased the annealing to 52 degrees Celsius. Still nothing. I tried increasing the concentration of gDNA. Still nothing. Perhaps it is the quality of the gDNA. Ideas?
3) Lastly, for one of the samples, I am getting a strong single band that is 100 – 150 bp longer than anticipated! I don’t know what could be causing this, but the band sixe is only longer in the gDNA, not the cDNA! What could this be? I have tried lowering the annealing and increasing the annealing, both resulting in the exact same band size that is just too big! Help!
I have checked the TM for all oligos, and they should be within range of the annealing. Should I mess with the number of annealing cycles, the extension time, etc?
Here is my PCR info--
Denature: 95 C – 30 sec
Anneal: (see above) – 30 sec x 35
Extend: 72 C – 30 sec