cloning problems

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Nov 10 11:46:08 EST 1998


In article <36485255.43925A4D at gengenp.rug.ac.be>, Esbjorn Fiers
<esfie at gengenp.rug.ac.be> writes
>Dear all,
>
>I ve been trying to do a simple cloning
>(fragment out of one plasmid into
>another) for some time already but
>things keep on going wrong. I ve got
>some questions now that will maybe help
>me to remediate my problems.
>
>1. Is it necessary to inactivate the
>restriction enzymes after the digest,
>before electrophoresis?

In simple terms no but if you are purifying a band from a gel than that
will need to be cleaned up away from contaminants in the agarose i.e.
inhibitors of ligation etc

>2. What is the equation for determining
>the composition of a ligation? I mean an
>equation that gives me the quantity of
>each fragment (in relation to its
>length) that should be added.

Molar ration of insert to vector of around 4:1 i.e for 1ug of a 3kb
plasmid use 4ug of a 3kb insert. 

>3. What is the correct composition of an
>selective blue white screening agar
>plate? I use X-gal, IPTG and
>carbeniceline in an LB medium but I have
>doubts the relative quantities. 

Per 400ml media I use 200ul 40mg/ml Xgal, 400ul 0.5M IPTG. Add your
antibiotic as required - not sure for carbenicillin as I never use it.


>Can you
>store a solution of X-gal in the
>refrigerator?

We keep ours in the dark in a freezer stored in NN dimethylformamide as
a 40mg/ml solution. 

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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