Q:QuickChange and deletions and native Pfu and cloned ....

[hadi]

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....

Regards,
hadi 

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). 
> Regards,
> V.
> 
> -- 
> Vladimir Svetlov
> McArdle Lab for Cancer Research
> Dept. Oncology
> UW-Madison
> 1400 University Ave.
> Madison, WI 53706
>