Tm and PCR optimization

rhubner at molbiol.ox.ac.uk rhubner at molbiol.ox.ac.uk
Mon Feb 14 05:52:25 EST 1994


Hi there everyone!
 I have a few questions about Tm and PCR optimization.
 I used the 2+4 rule to calculate an approximative Tm and checked this one 
with the program PRIMER 0.5 (I suppose - probably wrong-) that this program 
uses the (long) DNA formula with + 16.6 [Na+] etc...  I just learned that this 
is the formula used in OLIGO (a commercial PC product) with -675/length of 
primer. 
 Now, if you want to give thermostable pols, such as Pfu, a go the salt 
concentration is lower (say from 0.050-> 0.010) and this would lead to a 
correction of about 12oC with the above formula. PRIMER did calculate more
than 10oC lower Tm in this case... Stratagene indicate that a "minor" 
correction of Tm could be done - in my eyes 10oC is rather huge! What's wrong?
 Also, most people seem to NOT correct for salt concentration when using Pfu 
(limited literature survey!) I really would like advice on this...

Thanks so much.
  Roland


ps. For those who remember the quick exchange on Dynazyme a few months back, I 
announced that somebody here used successful TA cloning on the product 
(whereas it was announced to yield blunt-ends). I recently saw that "if 
blunt-ends are desired, the final primer extension time should not exceed 
5min. Otherwise, if an adenine is desired at the 3' end of the primer 
extension product, the primer extension time should be at least 15min."
   
 




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