PCRs no longer working. HELP!
gray at oci.utoronto.ca
Wed Mar 29 11:17:34 EST 1995
Have you checked/calibrated the PCR machine to make sure it is still ramping
On 19 Mar 1995, Shahram Mori wrote:
> 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.
> Shahram Mori _/\_
> Program in Molecular Biology _\ /_
> Dept. of chemistry and Biochemistry Box 3C \_ _/
> NMSU Las Cruces NM ||
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