Hi there,
I'm preparing my DNA samples for Pyrosequencing outside of my country so i'm wondering about concentration and volume that should be been in DNA samples before I send them?
Thanks in advance.
- ask the sequencing facility instead of asking here
- pyrosequencing is rarely done now a days, is this what you want to do or just with pyrosequencing you mean any NGS?
Pyrosequencing is a real-time sequencing technique based on the detection of released pyrophosphate during DNA elongation. This procedure starts once the PCR products are converted into single-stranded DNA (ssDNA) fragments and one strand is isolated (usually through labeling with biotin) for use as template in the Pyrosequencing reaction. To initially optimize the PCR amplification of the target DNA (without the addition of the universal biotinylated M13 primer to label the product), prepare a PCR mix in a final volume of 20–40 μl, according to standard PCR protocols, as follows: 25 ng of genomic DNA, forward and reverse amplification primers at final concentrations of 0.2–0.5 mM each, 1 × PCR buffer, 1.25 mM Mg2+, 0.2 mM of dNTPs and 1 U of Taq polymerase.
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