mutagenesis kit

Robert J. Nelson Robert.Nelson at ed.ac.uk
Thu Sep 6 05:30:51 EST 2001


Hi Michael,

Yes, this method of site-directed mutagenesis does PCR around the whole
plasmid, so it does have its limitations as to the size of the plasmid you
can mutate (although I'm not sure what this limit is.  I think we've done up
to ~10kb plasmids in the lab).  The method generates a nicked circular
plasmid and it is this that you transform with.

Pulled from the Stratagene website:
"The QuikChange site-directed mutagenesis procedure starts with a
supercoiled, dsDNA vector with an insert of interest and two synthetic
oligonucleotide primers containing the desired mutation. The oligonucleotide
primers, each complementary to opposite strands of the vector, are extended
during temperature cycling by PfuTurbo DNA polymerase. On incorporation of
the oligonucleotide primers, a mutated plasmid containing staggered nicks is
generated."

In your method, do you generate a linear PCR product that you then clone?
I've tried an overlapping PCR method similar to that with little success.
Strangely, I could get the 2 half reactions to work fine, but could never
get the final full length product.

I've also used a method based on the QuikChange protocol that requires only
one primer for each mutagenic site and can generate multiple specific
mutations in one go (although with considerably less efficiency than the
original authors):

Sawano, A. & Miyawaki, A. (2000) Nucl. Acids Res. 28: e78

Regards,

bob


---
> Dear Robert,
> do you mean you PCR around the circumerence of the plasmid,
> using the mutagenic primer pair and then transform with linear DNA?  Or do
> you cut and ligate?
> 
> I have been using a kind of two stage PCR reaction using 4 primers, two
> of which overlap:
> 
> ---->             <----
>                    ---->              <----
> 
> . . . if you see what I mean.  Regards, Mike.
> 
> 
> On 5 Sep 2001, Robert J. Nelson wrote:
> 
>> Hi there,
>> 
>> The Stratagene Quikchange PCR-based method has been extensively in our lab
>> for site-directed mutagenesis with great success.  We use their method, but
>> don't buy the kit because it's quite expensive.
>> 
>> All you need to do is a PCR with Pfu and complementary oligos spanning your
>> mutagenic site, Dpn I digest away the methylated template DNA, then
>> transform your bugs (you don't even need fancy ones that Stratagene suggest
>> - standard lab host strains can handle the nick repair).  Pick clones the
>> following day, miniprep and hey presto!
>> 
>> In my hands I get about 50 - 75% of transformants with the mutation
>> correctly incorporated (analysed by sequencing), the other 25 - 50% being
>> the original template and occasionally a plasmid with the mutagenic region
>> duplicated.
>> 
>> Hope this helps,
>> 
>> bob
>> 
>> 
>>> Dear all:
>>> 
>>> It has been a while since I used the promega M13-based mutagenesis kit
>>> back in 1994. Does anyone use more advanced commercial kit for in vitro
>>> mutagenesis? I found Clonetech " Transformer" and Strategene
>>> "Quickchange" kits. Welcome any users opinion on these products. Thanks.
>> 
>> ---
>> 
> 

---




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