Selection of recombinants
among the transformants by negative
selection
Colonies are randomly chosen from the ampicillin-agar plate,
and each colony is two times used:
-
to inoculate ampicillin containing medium (A),
-
to inoculate ampicillin-and-tetracyclin containing
medium (B).
Cells from colonies growing in A but not in B must be from
recombinant clones,
since most likely by the insertion of a Lambda DNA fragment,
the tetracyclin resistance gene is disrupted.
An easy way to process many colonies at once is to use
24-wells plates:
in two plates, bacteria from 24 colonies are grown in 1.5 ml
cultures.
After overnight growing the result could be something like
this: left: plate A, right: plate B
note:
Corresponding wells (e.g. the top left well on the left and the top
left well on the right) have been inoculated with the same colony!
Growth (turbid medium) should be seen in all wells with
amp-medium (left),
and there may be many clear (= non-growth) wells with amp+tet medium (right).
The cultures in the left plate will be further processed when
the same colony did not show growth in the right plate, since they will
be the recombinant clones..
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