PCRs no longer working. HELP!

[Andre Hamel]

Try following:

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 
adding template).

4/  Preheat thermocycler to 95*C & hold at 95 until rx'n tubes added 
(this equals hot start).

cheers,



                            ********************
  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
>> 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 
>> 
>>