cloning problems

Esbjorn Fiers esfie at gengenp.rug.ac.be
Tue Nov 10 09:48:54 EST 1998


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.






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