Bio-Rad Microbial Culturing Module - Manual (Page 8)

Preparation of Solid Media
Preparation of Ampicillin
Ampicillin is an antibiotic used to selectively eliminate bacteria that have not been transformed
with plasmids containing an ampicillin resistance gene. Ampicillin is shipped freeze dried in a
small vial containing 30 mg ampicillin. With a sterile pipet, add 3 ml of sterile water directly to
the vial to rehydrate the antibiotic to make a 10 mg/ml or 200x solution. Ampicillin is used at a
final concentration of 50 µg/ml in both LB-Ampicillin broth and LB-Ampicillin agar. Ampicillin
should be stored at –20°C and is good for 1 year.
Preparation of LB Agar
This protocol is used to prepare solid LB agar media for the growth of bacteria. Ideally agar
plates should be prepared at least two days before required and left out at room temperature
for two days and then refrigerated until they are to be used. Two days on the bench top allows
the agar to dry out and more readily take up the liquid transformation solution.
To prepare 500 ml LB agar, add the entire contents of the LB nutrient agar packet to 500 ml of
distilled water in a 1 L or larger Erlenmeyer flask. Swirl to dissolve the agar, or add a magnetic
stir bar to the flask and stir on a stir plate. A stir bar will also aid in mixing the solution completely
once sterilization is complete. Autoclave LB agar on wet cycle for 30 min. Once the autoclave
cycle is complete, check the solution to ensure that the agar is all dissolved. Alternatively if no
autoclave is available, heat the agar solution to boiling in a microwave. Repeat heating and
swirling about three times using a reduced power setting on the microwave to reduce
evaporation and boiling over until all the agar is dissolved (i.e. no more clear specks swirl
around). Be sure to wear appropriate protective equipment and be careful to allow the flask to
cool slightly before swirling so that the hot medium does not boil over onto your hand. When
the agar is dissolved, allow the LB nutrient agar to cool so that the outside of the flask is just
comfortable to hold (50°C). A water bath set at 50°C is useful for this step. While the agar is
cooling, label the plates. Be careful not to let the agar cool so much that it begins to solidify.
LB
NUTRIENT
AGAR
4
Add water Add agar
packet
Swirl
Microwave
to boiling