cloning problems

Rich Dudley rdudley+ at pitt.edu
Wed Nov 11 08:21:13 EST 1998


Esbjorn Fiers wrote:

> 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?
> 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.
> 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. Can you
> store a solution of X-gal in the
> refrigerator?
>
> Thanks in advance.
>
> Esbjorn Fiers.

Just out of curiosity, have you tried finding a copy of "Molecular
Cloning: A Laboratory Manual" by Sambrook, Fritsch and Maniatis?
Sounbds like it would be a great help to you.

rich

--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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