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Real-Time PCR using ABI Fast Sybr Green chemistry


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#1 Harvester

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Posted 13 December 2011 - 07:35 AM

Hey people,
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?
Thanks!

GP

#2 chabraha

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Posted 14 December 2011 - 03:16 PM

I have used the roche kit with good success............your initial denaturation step seems short..............try 5 min.........then do a three step PCR 10sec @ 95, 5-20sec @ 60 (or whatever your primers dictate), and 5-35sec (whatever your expected product size dictates @ 72.................acquire at the end of the 72 degree step.
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#3 Harvester

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Posted 15 December 2011 - 04:22 AM

Thank you chabraha for your reply. I am going to test your suggestions.




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