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?
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?