Purifying 5' phosphorylated primers for mutagenesis

apu via methods%40net.bio.net (by bt.arpita from gmail.com)
Sat Nov 28 06:59:25 EST 2009


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


More information about the Methods mailing list