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

    Then they plot linear Rn versus cycle and determine the cycle number that corresponds to ¼ 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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