Prerequisite Protocols

• Agrobacterium Heat Shock Transformation

Materials

• Taq 5x Master Mix
• Forward PCR Primer Working Stock 10uM
• Reverse PCR Primer Working Stock 10uM
• Sterile Distilled Water
• Agrobacterium tumefaciens GV3101 Colony Plate with well defined colonies
• LBKGR Liquid Media
• Sterile 2mL Round Bottom Tubes
• Sterile 0.2mL PCR Tubes
• Pipette (2.5uL, 20uL, 200uL)
• Purple Loading Dye 6x

Equipment

• PCR Machine
• Thermoshaker set to 28C 1000rpm
• Vortexer

Step By Step

1. Decide how many colonies you have or wish to screen and write down this number. Obtain that many sterile 2mL Round Bottom tubes. Add one extra tube for the negative control tube which will receive any bacteria.
2. Dispense 200uL of LBKGR Liquid Media with appropriate antibiotics added into each 2mL Round Bottom Tube.