Prerequisite Protocols

• none

Materials

• Escherichia coli NEB Turbo
• LB Broth (Miller Mod)
• TSS Buffer
• Sterile 2mL Round Bottom Microcentrifuge Tube
• 125mL Erlenmeyer Flask with foam stopper

Equipment

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

Step By Step

1. Streak a sample of E. coli cells onto LB Agar (Miller Mod) and incubate overnight at 37C.
2. The following day, select one colony with a sterile pipette tip or toothpick and place it in 20mL of LB Broth (Miller Mod) in a 125mL Erlenmeyer flask. shake the flask overnight at 37C, 250rpm.
3. The following day, dispense 1mL of the overnight culture into a sterile 2mL microcentrifuge tube on ice.
4. Spin down the cells for 15 seconds at 10,000G to gently pellet.
5. Decant the supernatant and add another 1mL of cells to the same tube.