Help! Separating 2 PCR products.

Dharsha 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 
size.  

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 
bands
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.

Dharsha



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