Rep (1) : Any Problems With Oligo? s??

martin LEACH leach at bu.edu
Fri Apr 5 08:05:02 EST 1996


Did you run your oligos out on a TLC plate (e.g. USB Surepure system) to see
how homogenous your oligo batch was?

Martin

Mr SHIRE David  (Tel 61.39.96.00) (David.SHIRE at TLS1.elfsanofi.fr) wrote:
: >  
: >  Fellow Bionetters,
: >     Recently a researcher in our Institute TA cloned a problem PCR product
: >  to find several incorrect bases in the region added by his primers,
: > > currently in cloning hell, we immediately suspected that some of our
: > > pimers (from the same supplier) are wrong as well. Rather than dis the
: > > supplier, who happens to be in the midst of an ultra low price campaign, I
: > > 'll ask if anyone has had recent bad oligos from any source. What company
: > > did you use? I'll be trying to TA clone the bad PCR products to see where
: > > the error lies but often there is a large element of trust involved with
: > > oligo buying and no one has the time for bad oligos. I'd appreciate any
: > > input here.
: > > 
: > > regards,
: > > Ted Michelini
: > > Institute of Molecular Biology
: >  University of Oregon
: >  tedm at darkwing.uoregon.edu
: >  
: >  
:  Hello Ted,
:  
: 	 Your problem is also one that is worrying me a lot. I am in charge of a service that  has been synthesizing and using oligos for 15 years and of course we update our machines every few years. With the latest machines the synthesis times are short and the amount of reagents used are low. So oligos are much cheaper to synthesize. Great ! This is why there is a big commercial battle going on between a multitude of suppliers, all using the same chemistry on more or less the same machines. But the downside 
is that the quality of the oligos obtained is pretty deplorable. On gels they look fine, but after PCR cloning and, as you may imagine, synthetic gene construction the number of constructs we have to sequence to get  correct sequences is very high. We are currently getting double insertions we have never had before, together with random point mutations in the synthetic sequences. All the reagents we use come from the manufacturer (a major and with absolutely no connection with my company) and the machines 
are tuned according to the manufacturer's instructions. Naturally, the supplier is of no help and we have started tinkering with the synthesis cycle because I think this is where the problem lies. Like you, I would be most interested in having other people's opinions because if the experience is general, then the machine merchants should look seriously into their synthesis cycles and maybe sacrifice a little cycle time and add some washes etc to improve the quality of the products.
:   In any case I would advise you, Ted, to tell the company you bought your oligos from about your problems, because they have to be aware about them in order to react. I can assure you that all the users here (and the sequencing service) are giving me hell !
:  
: David Shire,
: Sanofi Recherche,
: Centre de Labege,
: 31676 Labege, France
:  
: david.shire at tls1.elfsanofi.fr

--
.....          Martin Leach                Email:leach at bu.edu
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