Primer Problems

killer yeast vvsvetlov at utmem1.utmem.edu
Wed Jul 17 20:44:06 EST 1996


In article <hopkinsc.74.0009FDC9 at lincoln.ac.nz>, hopkinsc at lincoln.ac.nz
(Hopkins, Charlotte) wrote:

> I am hoping someone will be able to help me with a problem we are having with 
> our primers for the PCR.  We are following the instructions on reconsitution 
> but have been getting nothing working when using them in the PCR.  We went 
> back and tested the OD reading and it is very low (under half what it should 
> be).  It is getting to the stage we are doubting the synthesis of them. 

Hey, we had majority of our primers done locally by a person badly needing
brain transplant and good reading habits - on occasion I've got less than
half of the ordered primers in existance (note - half of the primers, not
half of every primer). So I've got more than enough experience with crappy
synthesis. First of all - OD does not mean much - unless you don't have it
at all - there can be shorter oligos that are precipitable and indeed UV
absorbant. If you doubt quality of your primers put them on the gel. PAGE
is fine but some consider it cumbersome just to see how bad you oligos
look, so we have been using 4% agarose (3% NuSieve, 1% SeaKem from FMC)
TBE e/phoresis to check the quality and if needed purify the oligos 20-70
bases long. It has lower resolution than PAGE but takes less time and
effort to set up and good enough to tell a band from smear and both of
them from nothingness. 

> Is anyone able to give me the name of another 
> company, that is similar in price, which you are pleased with their primers?
> Although we are in New Zealand, we are happy to send overseas for the primers 
> (we have to for Life Tech as well).

We ordered few times from National BioSciences, prices were rather
competitive although not dirt cheap and quality was superb. They have web
site at www.natbio.com and email nbi at natbio.com. In addition to synthesis
they provide free of charge an analysis of your primers with their native
program Oligo.n the only non-trivial oligonucleotide design software I've
seen. After that DNA contaminated dung that we were getting here dealing
with trained chemists was a welcomed change of pace. This is the place for
usual - "No affiliation with NBI or its parent/daughter/sibling/abusive
father company and no advertising is intended or implied, bla-bla-bla".
Again before reordering - check your oligos on the gel (normally 1/50 of
the 50 nmole synthesis on minigel should be enough to see this stuff - and
don't forget EtBr; you can use any commercial sequencing primer for
length/conc. comparison).
Regards,
Vladimir Svetlov

-- 
Of what use is a philosopher who does not hurt anybody's feelings?
                                Diogenes



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