High Ct Values, low amplification - (Aug/16/2007 )
I only started Realtime (sybr method) 2 weeks ago and have been getting high Ct values and low amplification (low fluorescence) of my template. I ran the reaction products on the gel and I see only 1 band. My melt curves look okay except for the fact that it has a low peak. Do you think it's possibly due to my template?
Any help would be great!
Trya mgCl2 titration, and a primer concentration. Also maybe try altering your annealing temp slightly.
getting a qPCR experiment up and running for the first time is not trivial. You might get lucky, but if you dont, ... much optimization will be in order.
Just have a look at the topics about PCR optimization. You should consider the efficiency of your primers. Very often, you will need to change the concnetrations of primers and/or template. Try doing serial dilutions of your template and your primers to find the optimal conditions.