Q:QuickChange and deletions and native Pfu and cloned ....
hadi at helix.nih.gov
Wed Jan 21 23:16:50 EST 1998
Hmmm, sounds good! Thanks for the update. So what are the guys from
Stratagene doing wrong...?
Concerning Pfu (to broaden this topic): Actually, I've used the kit only
once (I understand, that they sell native Pfu in the kit [it does really
exist?], however, the buffer in the booklet looked much more to me like
the one for cloned Pfu) and then switched to buying the enzyme separately
(cloned, of course..). I can't complain - if it works, it works and if it
doesn't work, it doesn't - no matter what Pfu. In my hands, fiddeling
around with the temperature profile has more effect than changing the
enzyme. But so far, I haven't done any deletions....
In article <svetlov-ya02408000R1901981101110001 at news.doit.wisc.edu>,
svetlov at oncology.wisc.edu says...
> To continue the topic - without any changes to the QuickChange protocol and
> using 62 nt long primers I've just finished making a 213 bp deletion.
> Efficiency was just below 50% (9 out 20 minipreps) which maybe attributed
> to the use of NEB's DpnI instead of either Stratagene or Boehringer (I use
> Boehringer enzymes whenever possible, but somebody wasted all the DpnI and
> we've got only NEB's enzyme in the on=site freezer). As I stressed before,
> the main factor allowing to use the "normal" QuickChange protocol is the
> design of oligos, where each "half" on either side of a large deletion
> considered to be annealing independently. The same protocol was used to
> generate smaller deletions using cloned Pfu polymerase and Pwo from
> Boehringer (as you know, kit's integrity is reversely proportional to the
> number of users, and native Pfu seems to be on the perpetual backorder, so
> if something is missing in the kit we just substitute the polymerase with
> whatever cat brings [but keep track of the appropriate buffers: I've been
> told (but never checked myself]), that even cloned and native Pfu's don't
> work optimally in each other's buffers).
> Vladimir Svetlov
> McArdle Lab for Cancer Research
> Dept. Oncology
> 1400 University Ave.
> Madison, WI 53706
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