As I dilute my qPCR template, the plateau of the curves also goes down. Does this mean my primers are too low, primers form dimers, DNA quality is poor, all of the above?
Hi,
What is qPCR template ? Master mix or cDNA?
If the sigmoidal curve flattens at a lower point I think it means the reagents such nucleotides have been used up earlier, so makes sense if you used a diluted master mix recipe.
You should check the melt curve for primer dimer.
Asif
Can you post a picture of your amplification curve and melting curve? As Asif mentioned the first reason would be the reagents are running out but there are other possibilities.
Dear Andrey
I concur with the other commentators
Another possibility is that with repeated heating over extended cycles you might, in conjunction with nominal inhibitors in your (unpurified ?) RT reaction be killing your qPCR polymerase and/or damaging, by deamination/depurination your cDNA undermining efficient replication
Regardless, failure to achieve a full plateau in your PCR/qPCr reaction normally implies some sort of poisoning/inhibition of your reaction
I would add therefore that if you are denaturing, keep your temp @ 94C; certainly no higher than 96C (unless your target region is > 60-70% GC)
Also, the concentration of your primer will affect the number of cyles necessary to attain your plateau: Primer concentrations anything from 2.5uM = 2.5pmoles/ul to 10uM are routinely used in qPCR (veering towards the lower end) so increasing your primer concentration within that range could help
- Badges
- Science topic