Prerequisite Protocols

• none

Materials

• Agrobacterium tumefaciens GV3101
• LB Broth (Miller Mod)
• TSS Buffer
• Sterile 2mL Round Bottom Microcentrifuge Tube
• Sterile 250mL Erlenmeyer Flask with foam stopper
• Sterile 50mL Tube

Equipment

• Orbital Shaker set at 200rpm at room temp (21 < t < 29)
• Static Incubator set to 28C
• Microcentrifuge capable of 10,000g
• Vortexer
• -20C Freezer (or -80C if you fancy)
• Ice Bucket

Step By Step

1. Streak a sample of Agrobacterium tumefaciens GV3101 cells onto LB Agar (Miller Mod) and incubate overnight at 28C.
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 28C 1000rpm.
3. The following day, dispense 500uL of the overnight culture into 100mL of LB Broth (Miller Mod) and shake at room temp at 200rpm overnight or until an optical density at 600nm of 0.500 is reached.