PCRs no longer working. HELP!

hodges bradley l segdoh at uxa.cso.uiuc.edu
Tue Mar 21 22:29:45 EST 1995


Maybe its your PCR machine.

On Thu, 16 Mar 1995, John Compton wrote:

> I have been working for 1 1/2 years on a project involving mapping and,
> now, sequencing of blood and buccal DNA samples from patients.  Initially,
> I experienced no problems amplifying these DNAs for microsatellite
> analysis and for template synthesis prior to sequencing.  Now, however, a
> dark cloud has appeared and only  specific, primarily buccal, DNAs will
> amplify.  I am using the same primers that worked before.  I have tried:
> new primer dilutions; synthesizing new lots of the old primers;  varying
> the amounts of DNA from 50 ng and up; altering the annealing temperature;
> varying the MgCl2 concentration; amplification using primers which would
> yield smaller (i.e. 300 bo rather than 800 bp) fragments; making new
> dilutions from the frozen stocks of DNAs; using all new reagents,
> including a different company's Taq; having other people in the lab set up
> the PCRs for me.  None of these attempts has altered my PCR success
> substantially.  
> 
> Does any one else have other suggestions, besides perhaps an extended vacation?
> 
> LR
> 
> -- 
> John G. Compton
> Lab of Skin Biology
> NIAMS, NIH Building 6 room 425
> 6 Center Dr.  MSC 2755
> Bethesda, MD 20892-2755
> jcomp at helix.nih.gov
> voice 301-496-7193, fax 301-402-2724 
> 
> 



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