ICSynBio:Agarose Gel
[u] Pouring LB Agar Plates [/u]
Introduction Plasmids can carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily isolate bacteria containing that plasmid from bacteria that do not contain it by artificial selection (i.e. growing the bacteria in the presence of the antibiotic).
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. The addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your own LB/agar plates with your antibiotic of interest.
[u] Equipment: [\u]
- Autoclave
- Water bath
- Pipetman
[\u] Reagents [\u]
- 37 g pre-mixed powder consisting of:
* 5 g peptone * 10.0 g peptone from casein * 10.0 g sodium chloride * 12.0 g agar-agar
- 1 L Sterile H2O
- Sterile plates of desired size
*60 mm x 15 mm plates holding 5-10 mL of agar ensures we can individually distinguish a maximum of ~100 colonies *100 mm x 15 mm plates which can hold 10 - 15 mL of agar for larger number of colonies
- Autoclavable flasks
- Sterile pipettes
- Ice bucket to hold antibiotic
- Antibiotic at 1000 x concentration dissolved in the appropriate liquid solvent.
