Prerequisite Protocols

• none

Materials

• Escherichia coli NEB Turbo Cells
• LB Broth (Miller Mod)
• TSS Buffer
• Sterile 2mL Round Bottom Microcentrifuge Tubes
• Sterile 250mL Erlenmeyer Flask with foam stopper
• Sterile 50mL Tube

Equipment

• Incubator-Shaker set at 37C 250rpm
• Static Incubator set to 37C
• Microcentrifuge capable of 10,000g
• Vortexer
• -20C Freezer (or -80C if you fancy)
• Ice Bucket

Step By Step

1. Streak a sample of Escherichia coli NEB Turbo cells onto LB Agar (Miller Mod) and incubate overnight at 37CC.
2. The following day, select one colony with a sterile pipette tip or toothpick and place it in 500uL of LB Broth (Miller Mod) in a 2mL Round Bottom Tube shake the flask overnight in a tube thermoshaker set to 37C 1000rpm. Alternatively, start a ~15mL volume of LB Broth (Miller Mod) in a 125mL Erlenmeyer flask with a foam stopper using a single colony from Step 1 and let it shake at 250rpm 37C overnight.
3. The following day, dispense 500uL of the overnight culture into 100mL of LB Broth (Miller Mod) and shake at 37C at 250rpm overnight or until an Optical Density at 600nm of 0.500 is reached. For Escherichia coli NEB Turbo cells in my lab it takes about 2hrs 15mins to reach the desired OD.