hot mutagenesis techniques

[Danuta Bois]

pnh at cockleberry.ncifcrf.gov (Paul N Hengen) wrote:



>Some years ago I had this same problem with the older verison of the `Altered
>Sites' kit. I screened numerous transformants from many different experiments
>by sequencing and never found the designed mutation. I gave up on it. FWIW,
>the only kit component I found necessary to buy the kit is the repair primer,
>which Promega will not give out the sequence of. Is anyone willing to disclose
>their own designed ampicillin repair primer or the one that's used in the kit?

>*******************************************************************************
>* Paul N. Hengen, Ph.D.                           /--------------------------/*
>* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
>* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
>* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
>* Frederick, Maryland 21702-1201 USA              /--------------------------/*
>* - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - *
>* - - -  Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - *
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Here are the sequences of oligos used in the Altered sites II kit from
Promega.  They're listed in the technical manual that came with the
kit:

1.Amp repair oligo: 5'-GTTGCCATTGCTGCAGGCATCGTGGTG-3'
2.Amp knockout oligo: 5'-GTTGCCATTGCGGCATCGTGGTGTCAC-3'
3.Tet repair oligo: 5'-GCCGGGCCTCTTGCGGGATATCGTCCA-3'
4.Tet knockout oligo: 5'-GCCGGGCCTCTTGCGGGCGTCCATTCC-3'
5.Cam repair oligo: 5'-CATTGCCATACGGAGTTCCGGATGAGC-3'
6.Cam knockout oligo: 5'-CATTGCCATACGGAACCGGATGAGC-3'

I hope this helps.

Gulf Coast Research & Ed.Ctr./U. of Florida
Bradenton, Florida