Real-Time PCR using ABI Fast Sybr Green chemistry
Posted 13 December 2011 - 07:35 AM
I just started setting up a ChIP protocol and this forum has been of great help. I have a "small" problem with my real-time PCR. Although end-point pcr is working fine, real-time PCR is not. I am using the ABI Fast Sybr Green and do a two-step PCR [95oC for 3secs and 60oC for 35secs] with an initial denaturing/taq activation step of 20secs at 95oC. The problem is that my Cts are over 45, which is not comparable with the end-point PCR results where 22 to 28 cycles are enough for different primer pairs to get the linear PCR phase.
I suspect that Fast Sybr Green needs a bit of a boost. Is anyone else using this kit to spare a tip? If not, could anyone suggest a sybr green kit that has been working for them?
Posted 14 December 2011 - 03:16 PM
Posted 15 December 2011 - 04:22 AM