oligo purification

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Mon Mar 15 19:43:39 EST 1993

>In article <jasons-120393211859 at mac3.medgen.uu.se>, jasons at bmc.uu.se (Jason
>E Stewart) wrote:
>> In article <1993Mar6.114601.11433 at iscsvax.uni.edu>,
>> jurgenson at iscsvax.uni.edu wrote:
>> > 
>> > Two questions: 1.   I have decided to get unpurified batches of pcr primers. 
>> > Will they need to be purified before use?  2. Does the mermaid kit from bio 101
>> > do this efficiently? The acrylamide method described in current protocols
>> > sounds very messy.
>> I feel necessary to add my $0.02 since I have been involved in the same
>> debate here at Med Gen.
>> Yes, you will most likely improve the efficiency of your PCR reactions
>> dramatically by purifying them! 
>> I can't believe that some of the scientists at our department order more
>> than 200 oligos/yr and then waste their grad students time by having them
>> run PCR without first running them on a gel! After months of frustration
>> they then say "there must have been something wrong with that primer
>> sequence that made it not work for PCR" - since that didn't use Oligo v4.0
>> either - they just "eyed" the primers! a Waste of time and valuable grad
>> student brain cells. To do a friend a favor I offered to run some primers
>> on a gel that he had by trying to get to work for three months ('cause he
>> thougth it sounded "too messy") only that the tubes were full of CRAP (i.e.
>> a so-called 35mer that wasn't longer than 20!!!). I have seen this time and
>> time again. 
>I suspect I will be one of the villains in your book when I confess
> that I never purify my oligos for PCR.  I think it depends on where
> you get your oligos from, and what you want to do with your PCR
> product.  We're up to oligo no. 103 in our lab, and for our purposes,
> we get by without doing anything to the oligos that we get from
> commercial suppliers.  We used to order oligos "trityl on", and
> then do an OPC column ourselves, but we don't even do this any more.
> Our oligos are for sequencing, cloning, and site directed mutagenesis.
> In most instances, we get by with probably with much less than max
> efficiency, but nonetheless we get what we need out of the experiment
> anyway.  
>My suggestion to the original questioner would be: if his oligo
> supplier is fairly dependable, to try the oligo first, (but not
> for THREE MONTHS ... sorry, but that particular student you mentioned
> obviously wasn't putting his valuable brain cells to good use
> in this case ...).  Then, if things don't work as expected
> (barring usual sources of error), take Jason's advice and verify
> that the oligo is of good quality.
>By the way, thanks for the detailed purification method.  I'm saving
> it for my notes, but I hope I don't have to use it!

I totally agree.  If you are having problems with primers: CHANGE your
supplier.  We have them made in house and NEVER need to purify for normal
purposes.  We purely dry down on a speedyvac to dryness, wash once with
~200ul water, redry and dissolve in water.  Spin in an eppendorf and use
the supernate.

Good Luck

Cheers. Klaus.

Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If you don't do: you rust"

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