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Old 01-28-2015, 01:23 PM   #7
zhengxiang he
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Location: NY

Join Date: Dec 2014
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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?
How about your experiment now?
We try several times. We start with 74k cells and can see clear cell pellet. But after we run the qPCR, we got very few signal on the qPCR and the curve is not good. Can you give us some suggestion?
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