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.

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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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