PCR hell

Chuck Dangler cad7 at psu.edu
Sun Jan 29 00:10:41 EST 1995


>In article <9501201302.AA10829 at compbio3.med.wayne.edu>, jeff at COMPBIO2.MED.WAYNE.EDU (Jeff Kramer) writes:
>> OK,
>> 
>>       So heres my problem.  (Sorry if my explitive in the subject heading 
>> offended anyone).  There was a grad student here in my lab who left last Fall.
>> He did about a million PCRs.  When I joined here, having never performed PCR
>> before in my short, chemistry major little life, I worked my way through the
>> learning curve, sacrificed all the apropriate cute little animals to the PCR
>> gods, and pretty much got everything to work.  Except for two primer sets.  One
>> of them, primers for human actin, apear to have wroked every time Jim (the
>> grad student) used them.  He used them at at least two different Ta's (60 and
>> 63) and with many different buffers.  They work fine.
>>       So, I come along, and they DO NOT WORK.  I have tried 50, 55, 57, 60,
>> 63, 65 and 68 C; 40, 60 and 120 seconds annealing time; 1, 2 and 5 min extention
 times (1.2 kb product); buffer pH's from 8.5 to 10; Mg conc from 0.5 mM to 3.5
>> mM (final conc); AmSulf conc from 5 mM to 20 mM, 5 and 10 % glycerol; 5 and 10 %
>> DMSO; perfect match; 5 % gelatin, 50, 100, 200 and 500 ng of primer/rxn; 2.4,
>> 24, 240 and 2400 ng genomic DNA (he used ~200); 100, 250, 500 uM and 1, 2.5, 5
>> and 10 mM final nTP conc; Tfl, Tth, taq and vent polymerases; hot start and no
>> hot start; two and three cycle amplifications; blah blah blah...

[This is the first time I have tried to reply to a newsgroup, so I hope this 
works.] First, I should say that I belong to the group that believes primers
can go "off".  However, my suggestion is to re-examine the Mg++ conc.  I think 
I have read elsewhere that the total load of target nucleic acid, dNTPs, and 
primers can impact on the required Mg++ conc.  To support this I have found 
that failed PCR reactions carrying high amounts of genomic DNA (sometimes I 
have been able to see obvious high molecular weight bands in the gels used to 
resolve PCR products) can be remedied by reducing the sample nucleic acid 
concentration, increasing the Mg++ conc, or some of both.  When I believe this 
is a problem, I usually suggest trying a range of 0.5 to 20 mM MgCl2.  Good 
luck.



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