Purifying 5' phosphorylated primers for mutagenesis

[Haley Lindsey]

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

[Ho Leung Ng]

For most cases, just desalted primers work most of the time. Using
purified primers can help if you use very long primers (50+ nt) or are
doing long insertions/deletions. For problematic primers, I like to
buy from Bioneer, which offers reverse phase purification for free.

ho

[Cathal Garvey]

I'm actually aiming to do a slight site-directed mutagenesis PCR reaction
shortly, I imagined it was as simple as doing a PCR with lower annealing
temperatures and a regular primer with a point mutation.. Am I mistaken?

What protocols do others on this list use for PCR-based mutagenesis? I'm
using KOD hotstart enzyme, which has exonuclease activity. It occurs to me
that it might inhibit any mismatches I might need for mutagenesis to occur,
should I be adding a solvent (such as DMSO) to inhibit exonuclease activity?

Thanks!
Cathal Garvey

[Duncan Clark]

Historians believe that in newspost
<mailman.373.12593...@net.bio.net> on Fri, 27 Nov 2009,
Cathal Garvey <cathal...@gmail.com> penned the following literary
masterpiece:

>I'm actually aiming to do a slight site-directed mutagenesis PCR reaction
>shortly, I imagined it was as simple as doing a PCR with lower annealing
>temperatures and a regular primer with a point mutation.. Am I mistaken?

No.

>
>What protocols do others on this list use for PCR-based mutagenesis? I'm
>using KOD hotstart enzyme, which has exonuclease activity.

Phusion also works nicely.

> It occurs to me
>that it might inhibit any mismatches I might need for mutagenesis to occur,
>should I be adding a solvent (such as DMSO) to inhibit exonuclease activity?

DMSO will do nothing to inactivate the proof-reading activity. You
actually want proof-reading activity, else your PCR product is going to
have errors where don't want them.

Look up Quikchange from Stratagene and use your KOD in your own homebrew
equivalent.

Duncan

--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

[apu]

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