Preparation of plates for bacteria:
Option for turning LB plates into LB-Amp plates
Dissolve 25 grams of LB mix in approximately 800 ml of water. Bring
the final volume to 1 liter. Pour this into a 2 liter flask and add
15 grams of Bactoagar (not agarose). Cover top with foil and autoclave
for 20 minutes.
After autoclaving, vigorously swirl the solution in the flask to mix molten
agar. Cool the solution to 50 oC - this is when you can
stand to hold the bottom of the flask for 10 to 20 seconds.
Add 100 mg of Ampicillin and swirl until it dissolves.
Commandeer a space that will hold 40 plates, unstacked. Pour plates
to a depth of approximately 3mm. If there are bubbles on the surface of
the agar, these can be remove by briefly flaming the surface with a bunsen
Leave the plates out at room temperature and unstaked. Wait at least
a few hours before stacking the plates and wait at least 24 hours before
putting the plates in th refrigerator. LABEL THE PLATES WITH THE
DATE AND NOTE THE PRESENCE OF AMP.
Store plates in the plastic sleeve in a refrigerator.
Add 200 ul of a 5 mg/ml stock of ampicillin; add drops of ampicillin
in several locations on the plate so that it is uniformly distributed.
Spread the solution around on the plate with a sterile glass rod and then
allow it to absorb into the plate. A stock of 25 or 50 mg/ml ampicillin
is in the -20 oC freezer, dilute the required amount to 5 mg/ml
Use of X-gal for blue/white screening.
Spread a mixture of 40 ul of 2% X-gal and 60 ul of water onto a plate,
let this diffuse into the plate for at least 1 hour
Just before plating the transformed cells, add 10 ul of 100 mM IPTG to
the cell mix.
X-gal should be made up in dimethyl formamide and stored in the freezer
- the tubes are often wrapped with foil since the X-gal is light sensitive.
In general, transformants that are white contain an insert in many of the
standard cloning vectors. If you are working with one of our plasmids
that has a ß-galactosidase reporter gene, you may find it useful
to use blue/white screeing to identify plasmids that contain the reporter.
Usually, a low level of activity is detected, presumably because the reporter
is transcribed in bacteria.
IPTG also stored in the freezer.