SYB green assay - (May/29/2005 )
SYB green assay in RT-qpcr:
The 3-steps cycle:
Denaturing: 15s 95C.
Annealing: 30s 60C.
Extension: 30s 70C.
Why should detection temperature be at least 3C lower than the Tm of amplicon when we want to have data collection after extension step?
SYBR green binds to double stranded DNA. You want your amplified product to be double stranded and so the temperature at which you aquire is just below the melting temperature of the amplified product. Are you confusing the Tm of the amplicon, which is likely to be above 72C, and the Tm of your primers ,60C?