Help???How to cip treatment with blunt-end vector???
dspinella at chugaibio.com
Tue Aug 26 10:11:23 EST 1997
Bipin Dalmia Writes:
> actually, i too always use CIP in the restriction digest directly
> instead of exchanging the buffer. CIP is a very robust enzyme and
> works well in all common restriction buffers. the main difference
> between restriction buffers is usually just the salt concentration and
> that really isn't much of a factor for CIP activity. here is my
> protocol (has worked every single time):
> after digesting your plasmid add 1 ul of CIP (per 20 ul of the
> original reaction)
> incubate at 37 C for 10 minutes
> add another 1 ul of CIP
> incubate at 50 C for 10 min.
> add 6X dye, run gel and purify your plasmid.
> i use this procedure even when the ends of my plasmid are
> non-compatible, to get rid of background from any single cut plasmid
> molecules that may be around.
> Bipin K. Dalmia, Ph.D.
> Protein Expression and Purification
> Pioneer Hi-Bred International, Inc.
> Johnston, Iowa 50131
This thread seems absurdly long given the triviality of the original
question, i.e. how to Cip treat a blunt-cut plasmid. My original
response to the posting was adapted virtually unchanged from Sambrook et
al. and yet to my surprise, seems to have provoked all kinds of
disagreement (some of it rather strident in tone). In the hope of
bringing this discussion to a conclusion, perhaps a final two cents may
clarify a few points:
1. Yes you CAN often do a Cip reaction by adding enzyme directly to a
restriction digest buffer. However, Cip likes ZnCl2 as a co-factor
which is not a common component in restriction buffers (yes Mildred, you
CAN just add it to the restriction buffer).
2. Cip may be a robust enzyme, but as anyone who has done a lot of
ligations into Cip treated vectors can attest, the reduction in the
frequency of re-ligated (non-recombinant) vector never reaches zero
(there are always a few colonies hanging out in that "no-insert" control
reaction) -- particularly when treating blunt or 5' recessed ends (as in
the original posting). If you are just trying to obtain a single
recombinant clone, it doesn't make much difference. However, if you are
trying to construct a library (which, btw, is what the original poster
relates in a private mailing that he is trying to do) or need to keep
backgrounds to a minimum, I still think you are better off precipitating
the DNA and performing the Cip reaction in the optimum buffer
3. The key issue with Cip to my mind is that it is hard to kill
completely with heat or even phenol extraction. Any residual Cip will
dephosphorylate the insert when it is added to the phosphatased vector
in the ligation reaction. Again, if you need a single clone it doesn't
much matter; but if you're trying to make a library, residual Cip will
reduce the number of recombinants you obtain. For this reason, a lot of
people prefer BAP or now Shrimp Alk.Phos. (which is more thermolabile).
I suppose you could also gel isolate the Cip'd vector to
electrophoretically separate it from any residual enzyme as Dr. Dalmia
describes above. This seems a bit labor intensive and of course one
always suffers some yield loss in the gel recovery, but I'm sure it will
work as stated.
If this thread has a common theme it seems to be that there is more than
one way to skin a cat and everyone has their own favorite variation on
the common protocols. So to each their own and if it works for you, go
Cheers -- D.G. Spinella
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