Getting primer dimers in RT-PCR on and off? - (Apr/24/2008 )
I am having trouble with my RT PCR primers, when i designed them i used the Oligo-analyser software to check for hairpins, self and hetero dimers and they were okay.
then i optimized the PCR conditions using standard PCR before trying them on the RT machine.
The funny and iriritating thing is:
1. first i ran three reps of negative control without DNA in RT machine and i get perfectly flat curves in the melt curve in 2 samples and a bump (indicating a primer dimer) in the other sample at abt 78 degs. I have obtained these results several times. why does this happen and yet its exactly the same thing just replicated?same reaction mix and all?
2. now today i got the shock of my life when i got not a just a little bump but high peaks at the same temp for all three control samples. i havent changed anything.
3. is my primer concentration too high? am using 0.5 ul of a 10um solution in 25 ul reactions.
Please help me understand why my results are not consistent esp the primer dimer thing. i have checked on agarose gel and its a true dimer abt 50bp.
How many cycles are you using, and when do the primer-dimers appear? With enough cycles, almost anything will show primer-dimers. Anything above 35 cycles or so is pretty meaningless without extremely careful preparation and excellent controls.
I am running 40 cycles, last week the Ct values for the controls were 37-38, while 2 out of the 3 reps were actually completely flat (i.e. the melt curve), this week the Ct value for the control was 25, and this time a very nice peak at 79 degs. this is the using the same protocol, but getting very different results.
would adding DMSO affect the results?