PCRs no longer working. HELP!

John Compton jcomp at helix.nih.gov
Thu Mar 16 14:21:42 EST 1995


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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