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Old 11-13-2014, 11:52 AM   #1
zhaolin
Junior Member
 
Location: Ann Arbor

Join Date: Nov 2014
Posts: 4
Default Rn/cycles: qPCR decided ATAC-seq library amplification

Hello, I have tried to set up a experiment following the ATAC-seq protocol. when I ran the qPCR side reaction to decide the cycle for library amplification, I met the problem.

The protocol uses Y-axis Rn 5000 RF as the threshold to decide the # of cycle since it is corresponded to of maximum fluorescent intensity.
In my experiment, I have tried cells # from 50,000 to 1,000,000 (the concentration of start PCR template DNA is 50 ng to 300 ng) but the maximum RF is around 200 RF(the amplification curve is fine). Should I only set 40 or 50 RF as threshold? (The correspond cycle is around 5.)
Has any one have similar issue? Could anyone help me here?
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