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Help Designing Primers

eric c. anderson anderson at pharmdec.wustl.edu
Tue Jul 26 10:30:31 EST 1994


In article <jpcd0-2507941501190001 at macr1-2.welc.cam.ac.uk>,
jpcd0 at mole.bio.cam.ac.uk (John Dixon) wrote:

> I need to design primers for sequencing from known sequence using a
> DyeDeoxy Terminator Cycle sequencing kit and analysis by an ABI automated
> sequencer. So far I have been using MacOligo to pick my primers for me,
> but to be honest I don't have a clue how it is selecting them and why it
> thinks some are better than others. I realize that you want minimal
> dimerization/hairpin and a unique 3'end at least. But can someone tell me
> where I can find a good guide to primer design explaining what attributes
> a good primer needs for different applications (ie Tm, length, GC richness
> etc etc - I dont have MacOligo Manual as we use a communal Networked copy
> of the program and I cant find the manual). 
> 
personally, i suggest picking them by hand.  none of the programs that i've
used (OLIGO 1.0, Geneworks, etc.) are very helpful for picking
autosequencing oligos because none of their algorithms were designed with
the machine in mind.  the user's manual and protocol manuals for the
different ABI kits have some suggestions for designing your oligos, and
those are the ones i tend to believe...more than any program.  i've had a
number of oligos work well for PCR but not for sequencing, with no obvious
reasons why.

also, one thing i've learned in the past 8 months of sequencing on our
machine is that your oligos and template CAN NEVER be too clean.  i EtOH
precipitate everything at least once now, and sometimes twice if i don't
trust the first time.  i don't know how clean your oligos are, if they get
gel purified then they're probably fine.  if like ours, they get cleaved
off and dried down, garbage and all, then they need some serious cleaning.

good luck,
eric
-- 

BIG AIR...

NOT BIG PANTS!!!
---------------------------------
eric c. anderson
anderson at pharmdec.wustl.edu
660 s. euclid box 8103
washington univ. school of medicine
dept of molecular biol. and pharmacology
st. loser, misery 63110



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