Intro
What...?
Available
Aim
Clone Example
E and B sites
Transformation
Ligation products
pGT4 in detail
Recomb in detail
Recombs Selection
Minipreps
Gel
Blot 1
Blotting
Labeling
Blot 2
Label Detection
Blot 3
Blot and gel
AE1
AE1 (cont.)
AE2
AE2 (cont.)
PCR

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..

The Simple Cloning Lab          Cloning Course