Help! Separating 2 PCR products.

Qianfa Chen qianfa at mgcheo.med.uottawa.ca
Thu Aug 28 10:45:26 EST 1997


Dharsha (mmm_dthurai at mednov1.auckland.ac.nz) wrote:
: 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

What about subcloning the mix of two bands into PCR2.1 (simple and easy) and you will 
have 50% of the clones that carry one of the bands.

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| Qianfa Chen  Ph.D.			   qianfa at mgcheo.med.uottawa.ca
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