Stock Solutions

LB Medium

1. Bring the following to 1000 mL with MQ dH20 in an autoclaveable bottle
   1. 20 g LB medium
2. Autoclave for 20 minutes on liquid settings
3. Once cool, add 100 mg ampicillin dissolved in MQ dH20 and dispensed through a 0.22 μm syringe filter

Sterile 50% Glycerol

1. Combine the following in an autoclaveable bottle
   1. 50 mL Glycerol (ρ = 1.26 g/ml)
   2. 50 mL MQ dH20
2. Autoclave for 20 min on liquid setting

Procedure

Overnight Culture

1. Under sterile conditions dispense ~8 ml LB media into a sterile round-bottom cell culture tube
2. Heat inoculation loop under flame until it glows red-hot and dip into (1) to cool
3. Immediately swab a single colony from an agar plate culture and transfer to (1)
4. Incubate overnight in 37 °C at 250 RPM circular agitation

Prepare Glycerol Stock

1. Under stirile conditions combine the following into sterile cryogenic tubes
   1. 750 μl Sterile 50% Glycerol (B)
   2. 750 μl Overnight culture
2. Store (1) in -80 °C freezer