PCRs no longer working. HELP!
hamel at cc.umanitoba.ca
Fri Mar 24 15:15:42 EST 1995
1/ Thaw reaction components 10 min @ 65*C (10x buffer & primers).
Include 10 mM NaOH in ALL primer stocks (helps minimize aggregates due to
prolonged storage in solution [freeze/thaw]) ... if primers fail over
time, don't discard, simply add 10 mM NaOH again! ... almost
ocunterintuitive, eh? ... but it WORKS! :^)
dNTPs need only be thawed on bench.
2/ After 10x buffer (ours has MgCl2 in it already) & primers are thawed
& heatedd @ 65*C -> reaction mixes MUST be set up on bench, this includes
addition of enzymes ... otherwise MgCl2 ppts out (can't see it, too
little to detect by eye ... compare making on ice versus on bench).
3/ THEN one can aliquot complete rx'n mixes, overlay with oil AND
finally put on ice after aliquoted ... after on ice for a couple of
minutes, addd template (ALIQUOTED rx'n mixes must be chilled before
4/ Preheat thermocycler to 95*C & hold at 95 until rx'n tubes added
(this equals hot start).
Andre Hamel email: hamel at cc.umanitoba.ca
Manitoba Government tel: 204/945-7630
Department of Agriculture FAX: 204/945-8062
Animal Health Centre
Winnipeg, Manitoba, CANADA
>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
>> Does any one else have other suggestions, besides perhaps an extended vacation?
>> 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
More information about the Methods