optimizing PCR reactions

szcooley at chip.ucdavis.edu szcooley at chip.ucdavis.edu
Tue Aug 23 11:05:15 EST 1994


Frederick Garbrecht (FRED at bmt.mcw.edu) wrote:
: I am curious as to how people go about optimizing PCR reaction 
: conditions when trying out new primer pairs.  One specific question I 
: have is, how reliable is the 'calculated' annealing temperature.  
: That is, say the calculated temperature is 60oC, and you don't get 
: specific bands in preliminary experiments, do you assume that some 
: other condition is not optimal (eg Mg++ concentration), or do you 
: begin to lower the annealing temperature in subsequent experiments.  
: Are there any hints as to how to begin to optimize, based on what you 
: see in initial experiments ( for example, no bands, or ladders, etc)? 
: My problem is that my thermal cycler is real sloooooow, so to run a 
: bunch of different temperature trials takes days, so I would like to 
: eliminate as much of that as possible up front.  I know there is 
: probably no easy way out, but I'd be very interested to hear how 
: you plan your strategies for optimization to get the right conditions 
: fast.  Thanks alot.



: Frederick C. Garbrecht
: fred at bmt.mcw.edu
: fgarbrec at post.its.mcw.edu
: Bone Marrow Transplant Program
: Medical College of Wisconsin
: phone 414 257 5053
: fax   414 257 7994

You might try "touchdown PCR". In this method it is not neccessary to 
optimize. You, in essence, run a ramp of the annealling temperature. The 
first cycles start at a high temp and subsequent cycles at lower and 
lower temps. The system works well for new primers where the problem may 
be something else in the reaction as well. Also you can "optimize" at the 
same time by making duplicate reactions and slipping a new tube into the 
machine after every couple of cycles. Depending on which ones work and 
which ones don't the optimal temp will be evident


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