PCR parameters - (Apr/23/2005 )
The protocol says: "All the assays run using the same universal thermal cycling parameters, for both TaqMan® or SYBR® Green I chemistry, as follow:
Hold 2 min 50 °C( optimal AmpErase® UNG activity )
Hold 10 min 95 °C( AmpliTaq Gold® DNA Polymeraseactivation and UNG inactivation)
For 40 cycles: 15 sec 95 °C 1 min 60 °C. "
What does it mean with “hold". What is the order of the program? Can different samples with different primer sets run together with the same program parameters? What makes it possible?
Can I know what product are you using?
Hold means, incubate. You can set you termal cycling profile as follow:
1) 50 °C 2 min
2) 95 °C 10 min
3) 95 °C 15 sec
4) 60 °C 1 min
goto step 3 repeat for 39 cycles.
Hold 2 min ( optimal AmpErase® UNG activity )
Hold 10 min ( AmpliTaq Gold® DNA Polymeraseactivation and UNG inactivation)
For 40 cycles: 15 sec 1 min . "
What suggested by the protocol is the general one.
In fact the temperature for RT step (50 °C 2 min), ativation step (95 °C 10 min) and denature step (95 °C 15 sec) are standard one. You can follow.
The annealing/extenssion temperature (60 °C 1 min) can be further optimised if you want to. You can run a gradient temperature ranging +/- 5 °C from your primer Tm. Select the best one.
Latter run a 10 fold serial dilution of your RNA ranging from 10E8 to10E1 to sonstruct a standard curve. You should see your PCR effeciency more then 95% and the slope of the curve -3.3 (ideal case)
I not recomanding you to run different sample and defferent primer set with the same programe. Because, different primer work in different efficency at defferent temperature.
That all I can say. Good luck
Do you know how many times i should run the PCR efficiency before i can accept it? Is that my product i have amplified or any other kind of standards with my target gene for making the standard? Should i do any inter and intra assay for each effiency test too?