3343 - Why is it difficult to quantify DNA using UV/Vis spectroscopy and fluorescence methods following EpiTect Bisulfite Conversion? How best do you quantify it?

Bisulfite converted DNA is difficult to quantify by spectroscopic methods.  No longer double stranded, PicoGreen and ethidium bromide cannot be used as they only bind reliably to ds DNA.  Single standed DNA uses a extinction coefficient factor of 40 to calculate DNA concentrations from OD 260 readings however the factors for each base is also different as U has a very different extinction coefficient factor than C.  This would therefore require a known sequence of C and an estimate of CpG.  However, the larger concern is the residual radical scavenger used to protect the DNA distorts the absorbance at 260 nm.  However, this chemical does not cause any inhibition of PCR.

QIAGEN recommends quantifying bisulfite treated DNA by real time PCR using methods developed for bisulfite treated DNA.  Highly concentrated bisulfite DNA (>100 ng/ul) can be roughly determine by spectophotometric measurement.

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