1 reply to this topic

[susanna]

Hi,

I wondered why in the sybr green master mix, a NTP mix,  with dUTP instead of dTTP, is used?
Does anybody knows this?

thanks,

susan

[laurequillo]

susanna, on Jun 9 2010, 10:38 AM, said:

Hi,

I wondered why in the sybr green master mix, a NTP mix,  with dUTP instead of dTTP, is used?
Does anybody knows this?

thanks,

susan

From the Applied Biosystem SYBR® Green PCR Master Mix
and RT-PCR protocol:

"When dUTP replaces dTTP as a dNTP substrate in PCR and the
method described below is used, AmpErase UNG treatment can
prevent the reamplification of carryover PCR products in subsequent
experiments Sninsky and Gelfand, pers. comm.) This method uses
enzymatic and chemical reactions analogous to the
restriction-modification and excision-repair systems of cells to degrade
specifically PCR products from previous PCR amplifications or to
degrade mis-primed, non-specific products produced prior to specific
amplifications, but not degrade native nucleic acid templates.
The method used to make PCR products susceptible to degradation
involves substituting dUTP for dTTP in the PCR mix and treating
subsequent PCR mixes with the enzyme uracil N-glycosylase (UNG,
EC 3.2.2-) prior to amplification (Longo et al., 1990)."