PCRs no longer working. HELP!

Shahram Mori smori at nmsu.edu
Sun Mar 19 18:31:23 EST 1995


John Compton (jcomp at helix.nih.gov) 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 
John take the Vacation Now and after you come back try seeing if your 
template is OK. What might have happened is that the template MIGHT be a 
little degraded thereby losing the primer sites. If it has worked before
try to check and see if there is anything (like changing the conc. of 
primer, dNTP's, enz etc... ) that is different now compared to then.
I wish you luck.
Cheers
 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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