Help???How to cip treatment with blunt-end vector???

[bipin k. dalmia]

On 21 Aug 1997 13:22:03 -0700, dspinella at chugaibio.com (Dom Spinella)
wrote:

>C. Rodemer (in a triumph of modesty and taste) writes:
>> I cut my PBluescript with EcoRI and didnt precipitate anything.I just
>> added buffer for CIP and some Units CIP .Then incubate at 37C. 
>> 
>You're a cloning stud!  However, since the original poster didn't say
>what restriction enzyme he was using for blunting, I thought it best to
>send him the "long" procedure since Cip isn't always fully active in all
>restriction enzyme buffers.

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.

bip 


Bipin K. Dalmia, Ph.D.
Protein Expression and Purification
Pioneer Hi-Bred International, Inc.
Johnston, Iowa 50131

dalmiabk at phibred.com