Bio-Rad Microbial Culturing Module - Manual (Page 7)

Preparation of Ampicillin Broth
This protocol is used to prepare liquid LB broth with ampicillin which will select for the growth of
bacteria transformed with plasmids containing an ampicillin resistance gene. The final
concentration of ampicillin in LB-Ampicillin broth should be 50 µg/ml. To make 25 mls of LB
broth containing 50 µg/ml ampicillin, add 125 µl of 10 mg/ml ampicillin stock. Ensure LB broth is
cooled to room temperature before adding ampicillin, excessive heat will degrade the antibiotic.
LB broth containing antibiotics should be stored at 4°C and is good for one month.
Starting Minipreps
This protocol is used to grow small cultures of bacteria in LB broth with ampicillin which will
select for the growth of bacteria transformed with plasmids containing an ampicillin resistance
gene. The small cultures are often processed to isolate plasmid DNA for experiments and
activities in molecular biology.
1. Prepare LB-Ampicillin broth as described above.
2. Label an appropriate number of 15 ml culture tubes.
3. Using sterile technique, pipet 5 ml of LB-Ampicillin broth into each 15 ml culture tube.
4. One day prior to the next lab session, use a sterile pipet tip or sterile loop to pick a single
colony and inoculate an LB-Ampicillin culture tube. A sterile pipet tip is often better than a
sterile loop to pick single colonies. Repeat for each miniprep culture from individual
colonies.
Note: Occasionally satellite colonies may grow so it is important to pick the large individual
colonies instead of the tiny colonies surrounding larger colonies. Be sure that a single colony is
picked, otherwise you may isolate multiple plasmids from your miniprep and these cannot be
sequenced.
5. Place the mini-prep cultures to grow in a shaking incubator or water bath at 37°C
overnight. The liquid cell culture should be shaken vigorously to provide a sufficient amount
of oxygen to the dividing cells. More vigorous shaking is better. Cells often may be grown at
room temperature with shaking but will require more time to grow.
6. Larger cultures can be inoculated by transferring 10 µl from the miniprep into a sterile flask
containing LB-Ampicillin and shaking.
7. Plasmid DNA can be isolated from minipreps using a plasmid isolation protocol such as the
Aurum Plasmid Mini kit (catalog #732-6400EDU).
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Swirl
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ampicillin