"

From 2012.igem.org

I. Inoculate Liquid Cultures for Transformation

• For MachI, ADP1, and PY79 cultures, inoculate 10 mL of LB with 100 uL of overnight cultures
• For EcNR1 and EcNR2ΔlacZ, inoculate 10 mL of LB with one colony from streaked plates (Note: these cultures were never used because they took too long to grow)
• Incubate at 32 ^oC for ~5 hours
• Check OD600 using 96 well plate and plate reader upstairs
  • RESULTS: ADP1 and PY79 cultures were at ~0.75, MachI had overgrown
• Add 5 mL of LB to MachI and return to incubator

II. Electroportation Transformation of ADP1 and PY79

• All steps performed on ice
• Aliquot 1 mL of cells each to 6 microcentrifuge tubes per strain
• Wash cells twice in 1 mL of cold sterile water from upstairs
• Resuspend cells in 50 uL of one of the following:
  • water (- control)
  • pBAV1K (+ control, 1 uL of 80 ng/mL in 50 uL water)
  • 1:1 1 (2 uL DNA in 50 uL)
  • 1:1 2 (2 uL DNA in 50 uL)
  • 1:1 3 (2 uL DNA in 50 uL)
  • 1:1 4 (2 uL DNA in 50 uL)
• Transfer each aliquot to a pre-chilled 2 mm cuvette (teal top)
• Electroportate using the GenePulser electroporatior with the following settings:

• • Voltage = 2500 V
  • Capacitance = 25 uF
  • Resistance = 200 Ω
• Immediately add 1 mL LB media, mix, and transfer as much as possible to a 5 mL culture tube
• Incubate at 32 ^oC with shaking for 60 minutes
• Transfer to microcentrifuge tubes and spin at 13000 rpm for 3 minutes to pellet cells
• Concentrate in ~50 uL of LB, resuspend
• Plate all samples on Kan(50 ug/mL)+IPTG+X-Gal plates with glass beads
• Incubate plates overnight at 34 ^oC

III. Electroportation Transformation of MachI

• Check OD600 again
  • RESULTS: It is under 0.8, proceed

• All steps performed on ice
• Aliquot 1 mL of cells each to 6 microcentrifuge tubes
• Wash cells twice in 1 mL of cold sterile water from upstairs
• Resuspend cells in 50 uL of one of the following:
  • water (- control)
  • pBAV1K (+ control, 1 uL of 80 ng/mL in 50 uL water)
  • 1:1 1 (2 uL DNA in 50 uL)
  • 1:1 2 (2 uL DNA in 50 uL)
  • 1:1 3 (2 uL DNA in 50 uL)
  • 1:1 4 (2 uL DNA in 50 uL)
• Transfer each aliquot to a pre-chilled 2 mm cuvette (teal top)
• Electroportate using the GenePulser electroporatior with the following settings:
  • Voltage = 2500 V
  • Capacitance = 25 uF
  • Resistance = 200 Ω
• Immediately add 1 mL LB media, mix, and transfer as much as possible to a 5 mL culture tube
• Incubate at 32 ^oC with shaking for 60 minutes
• Transfer to microcentrifuge tubes and spin at 13000 rpm for 3 minutes to pellet cells
• Concentrate in ~50 uL of LB, resuspend
• Plate all samples on Kan(50 ug/mL)+IPTG+X-Gal plates with glass beads
• Incubate plates overnight at 34 ^oC