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Stock Solutions
LB Medium
- Bring the following to 1000 mL with MQ dH20 in an autoclaveable bottle
- 20 g LB medium
- Autoclave for 20 minutes on liquid settings
- Once cool, add 100 mg ampicillin dissolved in MQ dH20 and dispensed through
a 0.22 μm syringe filter
Sterile 50% Glycerol
- Combine the following in an autoclaveable bottle
- 50 mL Glycerol (ρ = 1.26 g/ml)
- 50 mL MQ dH20
- Autoclave for 20 min on liquid setting
Procedure
Overnight Culture
- Under sterile conditions dispense ~8 ml LB media into a sterile round-bottom
cell culture tube
- Heat inoculation loop under flame until it glows red-hot and dip into (1) to
cool
- Immediately swab a single colony from an agar plate culture and transfer to
(1)
- Incubate overnight in 37 °C at 250 RPM circular agitation
Prepare Glycerol Stock
- Under stirile conditions combine the following into sterile cryogenic tubes
- 750 μl Sterile 50% Glycerol (B)
- 750 μl Overnight culture
- Store (1) in -80 °C freezer