Help! Separating 2 PCR products.
mmm_dthurai at mednov1.auckland.ac.nz
Wed Aug 27 16:33:28 EST 1997
I am trying to seperarte two PCR products, both 100-130 bp. They are
specific for my 2 (20 and 16 mer) primers. As I am trying to get the
Ig heavy chain rearragemnt (myeloma and ALL samples) PCRed up, it is
not unsuprising that I should get two products closely related in
I have tried to get just one band by:
optimising MgCl concentration-still two bands.
Increasing the temperature from "optimum" of 50 to 60 C-still two
Hot Start PCR-still two bands
Touch Down PCR-still two bands
Primer Titration-still two bands
The lower PCR band is ~120 and the upper band is ~130, so they are
separate but not easily separable. I have tried cutting out and
rePCRing the separate bands, but the lower band reappears. Using this
I have tried to get the lower band sequenced.
Please help me to try and get just a band form the PCR. Cutting the
bands is not easy or really reproducible, and thus is not a good thing
to use for screening our samples.
More information about the Methods