In vitro mutagenesis kit recommendation?

Steve L. Moseley moseley at u.washington.edu
Fri Nov 13 16:53:48 EST 1992


In article <BxMKJB.Lz8 at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu (the End) writes:
>From: jgraham at bronze.ucs.indiana.edu (the End)
>Subject: Re: In vitro mutagenesis kit recommendation?
>Date: Thu, 12 Nov 1992 22:45:10 GMT

>In <1dr98rINNcdb at usenet.INS.CWRU.Edu> ca566 at cleveland.Freenet.Edu (Michael Holloway) writes:


>>I apoligize if I'm lowering the intellectual level of this conference :-)
>>but I promised to post an inquiry to see if there are any favorite kits for
>>mutagenesis.  Is there any difference in the products?  Is there a
>>consensus on a favorite?

>>Thanks
>>Mike

>You are lowering the value of this forum. About half of the traffic here 
>is based on discussions of what is the best kit to "buy" rather than what
>is really important, the technical details of the procedures. 

>As with Exo III deletions, get yourself a copy of Maniatis, a few papers
>from the library (I recommend the Kunkel procedure) and yes, buy a few 
>salts from the stockroom. You don't need the high priced hand-holding
>from Biosham (TM). 

>Then come back with some intelligent questions, and I'm sure you will find
>more than enough help here.

>Cheers,

>Jim
>J. Graham
>Biology and Chemistry Departments
>Indiana University -Bloomington

Sorry Jim, but posts like yours aren't cause for any cheers. Your answer 
helps no one. Find another alt. or rec. group to vent your spleen and leave 
this group for those interested in learning and helping.

As to the original question, we've used the Kunkel procedure with salts we 
bought from the stockroom with some success. However, on the recommendation 
of a colleague whose intellectual level is lower than Dr. Graham's, we tried 
the T7-GEN in vitro mutagenesis kit from US Biochemical. We've been 
extremely pleased with its efficiency. A reference is Vandeyar et al., Gene 
65:129, of course those of us who use the K word probably don't know the way 
to the library. Mutant selection is based on methylation of the mutant 
strand, nicking of the original strand by restriction enzyme, and removal of 
the nicked original strand by ExoIII. It has worked very nicely for us. Most 
plaques are mutants with the kit, while in our hands, the Kunkel procedure 
efficiency was around 10%. 

 - Steve

 - Steve
__________________________________________________________________________
Steve L. Moseley                                  moseley at u.washington.edu
Microbiology SC-42                                Phone: (206) 543-2820
University of Washington                          FAX:   (206) 543-8297
Seattle, WA 98195



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