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Old 01-17-2018, 11:19 AM   #1
anish_dattani_zoo
Junior Member
 
Location: Oxford, UK

Join Date: Jan 2018
Posts: 1
Default qPCR for determining ATAC-seq amplification cycles?

Dear SeqAnswers,

From my understanding of the Buenrostro et al ATAC-seq papers, they PCR amplify transposed DNA fragments with 5 PCR cycles (using custom Nextera primers). After this initial 5 cycles of PCR, they do a qPCR citing the following reaction:

5 μl of previously PCR amplified DNA
4.41 μl Nuclease Free H2O
0.25 μl 25 μM Customized Nextera PCR Primer 1
0.25 μl 25 μM Customized Nextera PCR Primer 2
0.09 μl 100x SYBR Green I
5 μl NEBNext High-Fidelity 2x PCR Master Mix

1 cycle of 98C for 30 sec
20 cycles of 98C for 10 sec, 63C for 30 sec, 72C for 1 min

Then they plot linear Rn versus cycle and determine the cycle number that corresponds to of maximum fluorescent intensity.

I have never done this sort of qpcr before. Am I right in thinking that they add ROX to calculate the RN valuE - the Sybr green intensity value divided by the ROX? So whatever cycle number comes out from the above calculation, we do that many cycles for the next PCR?
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