AT to GC mutations

delisior at rnisd0.DNET.roche.com delisior at rnisd0.DNET.roche.com
Wed Apr 14 17:09:52 EST 1993


"jgraham at bronze.ucs.indiana.edu" "the End" writes:

>Robert,
>
>Have you had much success with the PCR randomizing mutagenesis ? I am following
>the Technique article and did not see any DMSO. Is that from the Yeast
>journal ? Are you getting 0.66% mutations per base (or 1 change in a 150 bp
>PCR product per round of 30 cycles) ? Are there hot spots or substitution
>biasis in your hands ?
>
>My intital attempts to get a product failed, as I did not size my PCR products
>well enough., and I subcloned a deletion of the intervening 100 bp.
>
>Thans much,
>
>Jim
>
>
Jim Grahm:

Details...............................Details...............................Deta
ils....

I am trying to isolate Ts/Cs mutants in yeast. My assay is qualitative not
quantitative and I sequence only that which is of interest to me. I have no
inclination nor time to quantitate  the mutation rate just to affix a number to
it. However, the Tecniques article does state that the frequency of base
mutations can be varied from 0.4% to 2% by selecting the appropiate conditions.
You will just have to work out the conditions for the results you need.

I do have a couple of brief comments regarding this protocol:

1) I have found in my hands that supercoiled DNA works as well if not better
than "linearized" DNA as template for Taq pol.

2) If you need to digest the PCR product in order to ligate it into the vector,
I have found that 6 bases after the recognition site is sufficient and
necessary.

Good Luck.

Bob D.


P.S. Both articles I cited use 10% DMSO. Prehaps you overlooked it.


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