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Old 11-13-2014, 01:19 PM   #2
obischof
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Location: france

Join Date: Jan 2011
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Quote:
Originally Posted by zhaolin View Post
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?
Very much depends on your PCR machine but we have similar data. On another note, if you put your amplified material on a gel or bioanalyzer do you observe a nuclesome-like pattern or rather a smear? We have trouble getting this defined nucleosomal pattern as shown in the protocol.
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