Bio-Rad Microbial Culturing Module - Manual (Page 9)

Preparation of LB-Ampicillin Agar
This protocol is used to prepare solid LB-ampicillin media which will select for the growth of
bacteria transformed with plasmids containing an ampicillin resistance gene. The final
concentration of ampicillin in LB-Ampicillin agar should be 50 µg/ml. To make 500 ml of LB
agar containing 50 µg/ml ampicillin, add 2.5 ml of 10 mg/ml ampicillin stock. Ensure LB agar is
cooled to 50°C before adding ampicillin, excessive heat will degrade the ampicillin. Swirl or use
the stir bar and a stir plate to mix the ampicillin into the agar taking care not to introduce bubbles.
Preparation of LB-Ampicillin IPTG Agar
This protocol is used to prepare solid LB-ampicillin IPTG (Isopropyl
b-D-1-Thiogalactopyranoside)
media which will select for the growth of bacteria transformed with plasmids containing an
ampicillin resistance gene. IPTG is used to increase protein espression induced by the lac
operon. The final concentration of ampicillin in LB-Ampicillin IPTG agar should be 50 µg/ml and
the final concentration of IPTG should be 0.2 mM. To make 500 ml of LB broth containing 50
µg/ml ampicillin and 0.2 mM IPTG, add 2.5 ml of 10 mg/ml ampicillin stock and 100 µl of 1M
IPTG. Ensure LB agar is cooled to 50°C before adding ampicillin and IPTG, excessive heat will
degrade the reagents. Swirl or use the stir bar and a stir plate to mix the ampicillin and IPTG
into the agar taking care not to introduce bubbles. IPTG is not included in this kit. A 1M stock of
IPTG is provided in Bio-Rad's Ligation and Transformation module (#166-5015EDU). Agar
plates containing IPTG are required to select colonies using the Ligation and Transformation
module.
Procedure for Pouring Plates
This protocol is used to prepare petri plates using the solid LB agar, LB-ampicillin agar or
LB-ampicillin IPTG agar media. Label the outside of the lower plate rather than the lid of the
plates that are to be prepared. Stack empty plates 4–8 high and starting with the bottom plate
lift the lid and the upper plates straight up and to the side with one hand and pour the LB agar
with the other. Fill the plate about one third to one half (~10 ml) with agar, replace the lid and
continue up the stack. Let the plates cool and solidify in this stacked configuration, do not disturb
them until the agar has solidified. Wipe any agar drips off of the sides of the plates. LB,
LB-Ampicillin, and LB-Ampicillin IPTG agar plates should be stored at 4°C enclosed in plastic
bags or plastic wrap to avoid drying out and are good for one month.
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