What kind of polimerase are you using? Is it used for other RT-PCR experiments and works fine? Maybe it is not stable enaugh to keep up the same rate of DNA replication.
Another possibility youd be that ou get multiple products from your PCR not beeing replicatet with the same rate since one is longer but since it is longer it gives a different signal?
yet another possibility is you are not replicating the full length of the or one of the templates resulting in this effect, since in higher cycles you get more shorter DNA pieces because they gradually accumulate compared to the full length product.
If you can give more information about reagents, if the primers are newly designed or from a paper. Did you try the standard program suggested by the software?
Hi Maulik, in Sybr Green qPCR chemistry, it usually happens when you have too much template as starting material in qPCR. Customize your template input in terms of its concentration and / or in volume or both, (Let's say for example take 2uL of cDNA in 10-25 ng concentration and primers less than or equal to 1 p mole / uL). These S shaped curves can be adjusted in post PCR run simply by changing the auto baseline mode to manual mode by a click and then, you must adjust the threshold for that certain gene(s) by opening delta Rn vs. cycle plot and linear mode view (do not adjust the threshold in log mode view). These curves will go away simply.
Regards....
@Jawwad Ahmad
Thank you for your reply. I am using 2 uL DNA (30 ng Concentration).
Primers & Probe i am using 1 uL out of 5 uM stock.
I did not change baseline mode, so i will try the same.
@Thomas
I am using Taq Polimerase from Sigma (5 U /uL).
I am working with only one product template at at time.
My gene target is 85 bp long. and using 45 cycle run.
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