Site-Directed Mutagenesis Kits
Jami Grossfield
commercially availbale kit would make this easier. I ordered the
stratagene ExSite kit- and their positive control doesn't even work! (yes
I have tried varying parameters, tech. support, etc.)
1) has anyone else had a problem with this kit?
2)does anyone know a good kit for deletion and point mutatation
site-directed mutagenesis kits? one they have used??
I know clontech makes one, and NEB- but I would like input from users
before buying another kit! please let me know if anyonw out there has
successfully used the ExSite kit from stratagene too!
thanks!
jami
the...@cc.umanitoba.ca
> Hi- I am trying to do site-directed mutagenesis and thought using a
> commercially availbale kit would make this easier. I ordered the
Jami;
We tried the Transformer kit from Clontech. Absolutely no success!! Zero!!
My advice is don't start with ds DNA, sart with ss DNA template if
possible. Chances for success are much greater.
Dave Boyd
--
Hey, hey, hey, hey! It was the DNA.
Hey, hey, hey, hey! That made me this way.
Karen.Straube-West
Hi- I am trying to do site-directed mutagenesis and thought using a
>commercially availbale kit would make this easier. I ordered the
>stratagene ExSite kit- and their positive control doesn't even work! (yes
>I have tried varying parameters, tech. support, etc.)
>
>1) has anyone else had a problem with this kit?
>2)does anyone know a good kit for deletion and point mutatation
>site-directed mutagenesis kits? one they have used??
>
>I know clontech makes one, and NEB- but I would like input from users
>before buying another kit! please let me know if anyonw out there has
>successfully used the ExSite kit from stratagene too!
>
>thanks!
>
>jami
We have been very pleased with the Muta-Gene kit from Bio-Rad. It takes a
little more work than some of the other kits, but our results have been
consistent. Another lab here obtained the kit components from us and has
had equally good success. One note: the ads state a 60-70% mutation rate,
but we have found its more like 20-30%, although one time I had 50%. Either
way, if you mini-prep 20 colonies, at least 4 should have your mutation.
There are actually two kits: one uses M13, the other uses a phagemid. We
use the M13 based kit. The ssDNA sequencing has also been a breeze for us,
a benefit of working with M13 over some of the ds plasmid sytems, IMHO.
There are probably faster approaches available (this will take about a week
once you have the constructs), but this approach has worked well for us.
Good luck!
Karen Straube-West
Dept. Pharmacology
SUNY Health Science Center
Syracuse, NY 13210
Chris Ferrer
> Hi- I am trying to do site-directed mutagenesis and thought using a
> commercially availbale kit would make this easier. I ordered the
> stratagene ExSite kit- and their positive control doesn't even work! (yes
> I have tried varying parameters, tech. support, etc.)
>
> 1) has anyone else had a problem with this kit?
> 2)does anyone know a good kit for deletion and point mutatation
> site-directed mutagenesis kits? one they have used??
>
> I know clontech makes one, and NEB- but I would like input from users
> before buying another kit! please let me know if anyonw out there has
> successfully used the ExSite kit from stratagene too!
>
> thanks!
>
> jami
Hi Jami,
During my previous post-doc at UT Southwestern, I used Promega's Altered
Sites Mutagenesis system (it is now Altered Sites II) with very good
results. Your DNA of interest is cloned into a phagemid, and ssDNA is
prepared (I recommend that a big batch of ssDNA is made at first since it
can serve as template for all your mutagenesis-it keeps well at -20). The
mutagenesis is performed on the ssDNA. Promega's secret weapon is the use
of a mutS strain post mutagesis that will not repair missmatched bases.
The mutagenized DNA is allowed to replicate in this strain before the DNA
is isolated and transformed into JM019. I usually picked 10-12 JM109
colonies, performed ds DNA sequencing (quick and dirty-nothing fancy) on 4
of the 10-12 transformants, and usually got 2 to all 4 to go my way. The
only time the mutagenesis failed was due to poor mutagenic oligo design
(the oligo had enough homology to another site on my template). I highly
recommend the kit. Promega now has versions of the kit that allow
expression of the mutagenized DNA.
Good luck,
Janet
Robert Horton
: In article <DEy70...@cf.ac.uk> Jami Grossfield wrote:
: >
: >Hi- I am trying to do site-directed mutagenesis and thought u
: >sing a
: >commercially availbale kit would make this easier.
: Hi, I am doing site directed mutagenesis as well. I don't use a kit but I do a PCR method as
: well that is pretty easy and works well, I get about 50 percent mutants.
: I use four primers, two normal PCR primers on the out sequences and then two on the inner, with
: the mutant, like this
: --rs------------- A ------x-------B
: =================================================== template
: ------x-------C --------rs--- D
: The first PCR is with A+C, B+D, that will produce two halves of your desired fragment, then
: take some, I use 1/10 of a 100ul reaction, of the first two and combine them into a second PCR
: step. Don't add more template and that should produce your full length.
: For more information, email me and i will send you a copy of my notes. But don't send here,
: send to my work email at:
: thanks
: andy
As much as you may find Andy's notes helpful, you may still want to look at
some of the original articles on the topic:
Higuchi R. Krummel B. Saiki RK.
A general method of in vitro preparation and specific mutagenesis of DNA
fragments: study of protein and DNA interactions.
Nucleic Acids Research. 16(15):7351-67, 1988 Aug 11
See also
Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R.
Site-directed mutagenesis by overlap extension using the polymerase
chain reaction
Gene 77:51-59, 1989.
The original reference for the variation on this technique using a single
mutagenic oligo is:
Sarkar G. Sommer SS.
The "megaprimer" method of site-directed mutagenesis.
Biotechniques. 8(4):404-7, 1990 Apr.
It is still considered good form to cite sources, even on the Net.
Robert M. Horton (PhD!) /\ "Crash programs fail because of the theory that
U of M Dermatology Dept || with nine women pregnant you get a baby a month"
Box 98 UMHC, 4-154 PWB /||\ -W. von Braun. Disclaimer: "Bob who?"
Minneapolis, MN 55455 ^^ hor...@biosci.cbs.umn.edu (612)625-8941
John K. Troyer
> Hi- I am trying to do site-directed mutagenesis and thought using a
> commercially availbale kit would make this easier. I ordered the
> stratagene ExSite kit- and their positive control doesn't even work! (yes
> I have tried varying parameters, tech. support, etc.)
>
> 1) has anyone else had a problem with this kit?
> 2)does anyone know a good kit for deletion and point mutatation
> site-directed mutagenesis kits? one they have used??
>
> I know clontech makes one, and NEB- but I would like input from users
> before buying another kit! please let me know if anyonw out there has
> successfully used the ExSite kit from stratagene too!
>
> thanks!
Jami:
I have been using the Promega kit (Altered Sites II) with good success.
In my experience, the major advantages of this kit are:
1) You can introduce multiple mutations simultaneously into the same cDNA
(I have introduced 3 mutations into one cDNA at the same time)
2) The kit works fine with double stranded DNA which greatly simplifies
the method since you do not have to make a single stranded phagemid
3) This kit is not PCR based. Therefore, you do not have to worry about
the fidelity problems inherent in many PCR methods.
Good luck!
Regards,
John
*****************************************
John K. Troyer, PhD
University of Maryland School of Medicine
Department of Biological Chemistry
(410) 706-7518
jtr...@umabnet.ab.umd.edu
*****************************************
Robert Horton
: Robert Horton (hor...@biosci.cbs.umn.edu) wrote:
: > ...
: > It is still considered good form to cite sources, even on the Net.
:
: Robert is right and I should have posted the sources. I was not
: trying to take any credit, merely trying to help a fellow scientist
: out. If my intentions were misconsrued, then I am sorry...
On re-reading my previous reply, I see it is more harshly worded than I
intended. Andy was being helpful, and I hope I haven't discouraged him
from speaking up in the future. Sorry.
I hope people realize that asking folks to cite sources for methods is
not *just* an ego thing. Referencing papers in this group (hopefully)
increases their chances of being cited in press, which helps people
justify the effort they spend developing methods. And the papers do
tend to go into greater detail than the posts. Besides that, being cited
gives you a warm, fuzzy feeling :)
But I promise to try to be nicer about it from now on.
Richard E. Showalter
>
> Hi- I am trying to do site-directed mutagenesis and thought using a
> >commercially availbale kit would make this easier. I ordered the
> >stratagene ExSite kit- and their positive control doesn't even work! (yes
> >I have tried varying parameters, tech. support, etc.)
> >
> >1) has anyone else had a problem with this kit?
> >2)does anyone know a good kit for deletion and point mutatation
> >site-directed mutagenesis kits? one they have used??
> >
> >I know clontech makes one, and NEB- but I would like input from users
> >before buying another kit! please let me know if anyonw out there has
> >successfully used the ExSite kit from stratagene too!
> >
> >thanks!
> >
>
I would have to second the Bio-Rad Muta-Gene kit. We have made our
expression vectors phagmids by placing an F1 ori in the plasmid. This way
you can make a series of mutants in a gene that you can already express
without having to do *any* recloning of the mutant construct, and, you can
do double or single stranded sequencing on the same plasmid to verify the
mutation. I am surprized to see such a low mutation frequency from the
above poster. I generaly get >80% mutation frequency. I only need to
sequence 1-2 isolates per mutation reaction to get the clone I want. I do
tend to make my mutation oligos long >30bp and I am very careful about the
primer to template ratios as recomended in the protocols. I have used it
recently to generate over 30 different single and multiple site AA
substitutions, deletions, insertions, restriction site modifications, you
name it. Works for me. YMMV.
Regards,
--
Richard E. Showalter "Hey Hey Hey Hey it was
Senior Associate Scientist the DNA. Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc. that made me this way."
sh...@agouron.com Queen-Shear Heart Attack
vip...@bsa.bris.ac.uk
based on the phosphorothioate method of Fritz Eckstein. I haven't yet
had a failure (lucky me!).
I don't know whether it's been updated since I bought mine. It requires
single strand DNA. The instructions are fairly detailed and easy to
follow.
Barry