PCR optimization

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Aug 23 16:04:18 EST 1994


Frederick Garbrecht writes:

> I am curious as to how people go about optimizing PCR reaction 
> conditions when trying out new primer pairs.  

I use NBI's Oligo program (no affiliation) and optimize all my primers
for one common set of conditions rather than the other way around.
Saves a lot of aggravation.

> One specific question I 
> have is, how reliable is the 'calculated' annealing temperature.  

Some methods are indexed to 1 M Na+ so you have to make an adjustment
(16.6 log[Na+]).  After that they all tend to agree within a few
degrees.  They are all 8-10 degrees low (at estimating Tm) 
because they ignore the Mg++.  Some methods have a built in adjustment
that takes care of this when they compute an optimal 'annealing' temp.
(as opposed to 'melting' temp.).  That's why some methods recommend
an annealing temperature above the Tm.

> 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.  

The trouble with adjusting Mg++ is that you're altering the primer Tm, the
activity of the nucleotides, and the stability of the template all at the same
time.  

> 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)? 

Ideally the primer design session will give a more specific
indication of what problem to address.  If template stability is pushing
the denaturation temperature, then changing Ta won't help; you need to
add formamide or some other denaturant.  If the problem is nonspecific
background, then I would normally know that I had only about 5 C of 
margin to raise the Ta; so if I don't suppress it on the second try,
it'll probably never work.  If the analysis indicated a potential for
non productive primer complexing, then higher primer concentrations
may be the answer. etc.  I've seen a lot less nonspecific background
since I started making the primers with lower stability on the 3' end.


> 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.

Since I started putting more effort into the design, most of my pairs 
work on the first try, and most of the rest on the second try.  The
few that still give a lot of trouble are usually related to some
special design problem that we knew about but tried to slide by.  We
never see primer dimers any more.  Before, I was spending a fortune
remaking primers by trial and error.

Good luck
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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