PCR DIG labeling kit and G+C-rich DNA

Steven L Goldberg steven.goldberg at bms.com
Fri Sep 15 12:12:54 EST 2000


I have been using Roche's PCR DIG labeling kit to make
hybridization probes.  My target DNA is very G+C rich (ca. 75%),
which may be the reason for the difficulty I have occasionally
have obtaining amplification and/or incorporation of the DIG
label.  Roche's technical services suggested diluting the DIG
label with dNTPs, which does help in some instances.  Other
times, I have to make new primer sets to my region of interest.
However, there are some cases where I cannot get any
amplification at all with the kit, although using other buffer
systems such as FailSafe gives me very good amplification.  Has
anyone else had the same experience and if so, how was this
problem overcome.  Is it possible to use a buffer different from
the one provided with the PCR DIG kit and still get incorporation
of the label?  Thanks for any advice.

Steve






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