Primer Problems

brett brett at BORCIM.WUSTL.EDU
Tue Jul 23 11:35:36 EST 1996


>In article <hopkinsc.74.0009FDC9 at lincoln.ac.nz>, 
>hopkinsc at lincoln.ac.nz
>(Hopkins, Charlotte) wrote:
>
>> I am hoping someone will be able to help me with a problem we are having with
>> our primers for the PCR.  We are following the instructions on reconsitution
>> but have been getting nothing working when using them in the PCR.  We went
>> back and tested the OD reading and it is very low (under half what it should
>> be).  It is getting to the stage we are doubting the synthesis of them.     
>> 
>
>
>   We have had good fast and inexpensive oligo supply from Beckman in 
>Sydney and from Bresatec in Adelaide.  Bresatec is currently the 
>cheaper of the two.  
>
>-- 
>Richard C. Nicholson, BSc PhD
>Senior Research Fellow
>Centre for Immunology
>St. Vincent's Hospital
>Sydney, Australia 2010


Check them on a PAGE to size them and estimate their concentration.
Additionally, clean them up with 5x butanol etractions (water-sat'd),
Reppt.
and resuspend. Finally, question the quality of the primer *design*. Consider
possible false primings, primer dimers, and incorrect Tm as potential problems.
These things are much more likely than some tech misprogramming an oligo
synthesizer, and should get you to some working primers faster than complaining
to the company. Also, I assume your PCR's are otherwise working? ie. you have
some (+) controls, especially for the template in question? Also, try another
enzyme. I recently had problems with KlentaqLA on one template, but no others.
In a second round of attempts it worked only in reactions that contained DMSO.
However, Vent worked great in parallel, even w/o the DMSO. Strange? You bet, but
at least I got my product. Good luck!



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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