theory beyond protocol

[wilczka]

Hello,
I am working on IQ5, biorad cycler, and I have some doubts - I use the same protocol type as recommended in manual and I've found out I do not understand it.
The protocol is:
1 Cycle - 95C - 1 min, 2 cycle 1 step 95C, 10s, 2 step - (my annealing temperature) - 30s, in this step the fluorescence is counted. The 2 cycle is repeated 40 times and that is it. Where is the template elongation?? How does this PCR work?

Thank you for any hints

[sunglassesatnight]

That's wierd. Maybe elongation occurs during the ramp up to 95? There really should be an explicit elongation step.

[fluori]

I use the same cycle program with the ABI 7500. Works fine. Although, my meltingcurves do have the same temperature even when the template is different.
My probe based single as well multiplex do not give any problems with this cycle program.

About the elongation step, see this discussion:
http: // www . protocol-online.org / biology-forums / posts / 26667 . html

Hope you'll understand it now