ChIP and qPCR - ChIP and qPCR (May/06/2008 )
Hi guys,
Can anyone help me? I'm doing qPCR on my ChIP samples and am having major difficulty. Using SYBR green and use the fast ChIP protocol.
Can anyone advise me on a good protocol to use?
Thanks H
Can anyone help me? I'm doing qPCR on my ChIP samples and am having major difficulty. Using SYBR green and use the fast ChIP protocol.
Can anyone advise me on a good protocol to use?
Thanks H
what difficulties are you having?
Well I have a protocol where I add 1.2uM of my primers, 10ng of my samples and then 15ul of the SYBR Green Matermix but I can't see any difference between my input and ip samples and I am only getting formation of product at around 30 cycles.
When I dilute my input by a factor of 10, I cannot see a 3 cycle difference to the original sample.
When I dilute my input by a factor of 10, I cannot see a 3 cycle difference to the original sample.
Have your previous chips worked?
One explanation of diluting the DNA down but not seeing a difference in Ct values is that the PCR may not be working efficiently. Have you done a standard curve on a dilution series of DNA to ensure your PCR is efficient?