Purifying 5' phosphorylated primers for mutagenesis (Haley)

Haley Lindsey via methods%40net.bio.net (by haleyl from u.washington.edu)
Mon Nov 30 13:30:24 EST 2009


Arpita: At the places I've looked (invitrogen, operon, and some others),
most of the expense was associated with PAGE or HPLC purification which
increased the price to 60 or 70 dollars per primer. For a phosphorylated 30
bp primer that has been desalted--which most of the responders indicated
should be sufficient for purification--the cost is more like 20-25 dollars.

Thanks to everyone who responded!

Haley

On Sun, Nov 29, 2009 at 9:04 AM, <methods-request from oat.bio.indiana.edu>wrote:

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>   1. Re: Purifying 5' phosphorylated primers for mutagenesis (apu)
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> ---------- Forwarded message ----------
> From: apu <bt.arpita from gmail.com>
> To: methods from net.bio.net
> Date: Sat, 28 Nov 2009 03:59:25 -0800 (PST)
> Subject: Re: Purifying 5' phosphorylated primers for mutagenesis
> On Nov 25, 3:12 am, Haley Lindsey <hal... from u.washington.edu> wrote:
> > I'm doing some site-directed mutagenesis with a kit from NEB. It is
> > recommended in the kit directions that the 5' phosphorylated primers used
> > for the reaction are purified either using RP-HPLC or PAGE.
> >
> > My issue is that these purification methods are *really* expensive. (I've
> > been getting my primers through Invitrogen, but I've also looked at a few
> > other places and it's the same story.) Has anyone had success using
> primers
> > purified another (less expensive) way or done the purification in house?
> >
> > Thank you,
> >
> > Haley, University of Washington
>
> Can you please tell me what are the costs you came across? Actually I
> have ordered some long primers from MWG through our institute's
> centralized facility, and got to know they are expensive. But how
> much? They are not telling it clearly.
>
> For single site SDM, purified primers are not necessary. HPSF is
> sufficient. I have done several of them with HPSF purified and 5'
> unphosphorylated primers.
>
> Best regards,
> Arpita
>
>
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