jchou at cgl.ucsf.edu
Wed Dec 11 18:55:23 EST 1996
vvsvetlov at utmem1.utmem.edu (Vladimir Svetlov) writes:
>In article <32AD5CF3.652D at ucdavis.edu>, jrwiliams at ucdavis.edu wrote:
>> I was wondering if anyone could recommend a good mutagenesis kit?
>> Promega and Stratagene both offer kits. Has anyone used these? Are they
>> any good? Thank's in advance for any info.
>> Jeff Williams
>> jrwilliams at ucdavis.edu
>Never used Stratagene kit. Promega's kit was OK but rather tedious with
>all those phage preps. Last couple of times I've used their protocol
>modified for use with ds template and got 7 out 8 and 2 out 8 clones
>sequenced with desired mutations (in the last case I believe that the
>mutagenic oligo was funked up during synthesis). DpnI based kit was used
>by some other people in the lab and worked like crap.
>Moral: Promega is quite reliable and in ds protocol is a breeze.
I've had good success with Promega's "Altered Sites" kit, which has the
option of double-stranded template denaturation as well as isolation
of single-stranded DNA. Using the ds protocol (no phage work necessary),
I was able to use 3 mutagenic oligos as well as their 2 drug-resistance
mutagenic oligos, and get all 5 site-directed mutations to work
simultaneously. (I think it was only about 1 in 8, but the other 7 in
8 usually usually had 2 of my 3 oligos work.)
| Joe Chou <jchou at socrates.ucsf.edu>
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