phosphatase question (library construction)

Alexander Kraev kraev at bc.biol.ethz.ch
Tue Mar 11 10:54:05 EST 1997


In article <Pine.SOL.3.95.970310123236.7409A-100000 at ascc.artsci.wustl.edu>,
 Alex Brands <abbrands at artsci.wustl.edu> wrote:

> I'm about to attempt construction of a cDNA library into a GAL4AD plasmid.
> I have the mRNA, I have the kit, and I have cells that are 2X10^8/ug, so
> I'm almost ready to go.  The problem is, I tried a ligation of the
> prepared vector, and I got some background.  Not much, but some.  It was
> prepared by cutting with EcoR1, CIP treatment, followed by gel
> purification, organic extraction, precipitation.  

Now, you seem to have performed a negative control, i.e. self ligation.  Have
you considered a positive control, i.e. how many clones you get with a test
insert (a single gel purified fragment)  in increasing amounts. If you are able
to get  20 fold more colonies with an insert than without  it  (due to
self ligation),
that must be enough for a library.  This also tells you which fraction of your
vector is available for ligation, and this is the way to test if more
phosphatase
helps or  not  (see below) 
> 
> My question is this; If I use more CIP, I could reduce the background, but 
> are there any possible detrimental effects of using too much CIP?
> How about SIP?

Well, with a good phosphatase what you achieve in a normal reaction can
hardly be improved.  And today's phosphatases are usually very good  in
terms of purity. In my hands, CIP (at 50 C) was better than SIP for blunt
and recessed
ends, and the same for protruding ends.  For quick cloning SIP has an
advantage of being thermally inactivatable,  but  since you must also ensure
complete cleavage for low background without gel purification of the vector,
the advantage of SIP, IMHO, is small, except for special cases. It was
introduced,
I think, as a result of complaints from people desperately trying to get rid of
of BAP/CIP without gel purification.

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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