Team:British Columbia/Protocols

1. E. coli Chemical Transformation

Protocol:

1. Add 2 uL of plasmid into thawed chemically competent cells.
2. Incubate for 10-15 minutes on ice.
3. Heat shock epitube at 42°C for 60 s.
4. Remove and put on ice for 3 minutes.
5. Recover with 400 uL of LB media.
6. Incubate in shaker at 37°C for 1-2 hours.
7. Plate 150 uL on selective plates, or spin down to increase concentration
8. Incubate plates at 27°C for 24 hours.

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2. Restriction Enzyme Digestion

Materials:

• 5 uL buffer
• 1 ug DNA
• 1 uL restriction enzyme 1
• 1 uL restriction enzyme 2
• up to 50 uL dH₂0

Protocol:

1. Incubate mixture at 37°C for 1-2 hours.
2. Heat inactivate by incubating at 65°C for 15 minutes.

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3. Ligation

Materials:

• 2 uL T4 ligation buffer(NEB)
• 2 uL plasmid
• 8 uL insert
• 7 uL H₂0
• 1 uL ligase

Protocol:

1. Combine reagents, adding ligase last
2. Leave mixture at room temperature for 1 hour.
3. Heat inactivate at 65°C for 10 minutes.

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4. E. coli Chemically Competent Cell Preparation

Materials:

• LB media
• 0.1 M CaCl₂
• 0.1 M CaCl₂ + 15% glycerol
• Liquid N₂

Keep these on ice for >30 minutes:

• 200 mL centrifuge tubes
• 50 mL Falcon tubes
• 0.5 mL microfuge tubes

Protocol:

1. Inoculate 100 mL overnight culture in LB at 30°C.
2. Transfer 1 mL overnight culture to 100 mL LB (or scale up) and incubate shaking at 30°C for 3-4 hours until OD₆₀₀ reaches 0.5-0.6.
3. Chill culture for 10 minutes on ice.
4. Spin culture for 10-15 minutes at 4000g 4°C.
5. Remove supernatant and resuspend with 30-40 mL of 0.1 M CaCl₂.
6. Incubate on ice for 30 minutes.
7. Spin culture down for 10 minutes at 4000g 4°C.
8. Remove supernatant and resuspend in 6 mL 0.1 M CaCl₂ + 15% glycerol.
9. Aliquot 50 uL in 0.5 mL microfuge tube and snap-freeze in liquid N_2.

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5. Electrocompetent Agrobacterium Cell Preparation

Prepare/wash thoroughly in advance:

• Autoclaved LB (3 x 1 L flasks, 1 x 50 mL flask)
• 10% glycerol (4 x 1 L flasks)
• Centrifuge bottles (6, autoclave with ddH₂0 inside)

Protocol:

1. Streak stock cells on LB plate and grow at 30°C overnight.
2. Use a single colony to inoculate a 5 mL LB culture, agitate at 30&#176C overnight. Put three uninoculated 1 L LB flasks into 30°C overnight, and 4 1 L 10% glycerol stocks into cold room overnight.
3. Use 1 mL starting culture to inoculate each of three warmed LB cultures, and agitate at 30°C for 5-6 hours, until OD₆₀₀ = 0.8-0.1.
4. Chill te three flasks on ice/water in the cold room for 30 minutes with some shaking.
5. Use six centrifuge bottles to spin down all of the cultures (500 mL in each bottle) at 4000 g at 4°C for 15 minutes. Remove as much supernatant as possible, as all of the salt is in the pellet at this point and the pellet will be packed tightly. Add small amount (1 mL) of 10% glycerol to cells to resuspend (be vortex in cold room) and combine into two centrifuge bottles.
6. Perform washes by resuspending cells in sterile cold 10% glycerol in the cold room (500 mL per bottle) and spin as before. Repeat twice more for three total washes.
7. During final wash, fill a large autoclave tray with ice and arrange approximately 100 Eppendorff tubes in a tray, lids open.
8. Remove the supernatant from the final wash of the cultures, and then resuspend cells in whatever liquid remains and aliquot 60 uL per Eppendorff tube. Put into liquid nitrogen immediately, then store in -80°C freezer.

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6. Agrobacterium Electroporation Protocol

Protocol:

1. Chill 1mm electroporation cuvette on ice for 10 minutes.
2. Thaw electrocompetent Agrobacterium on ice for 10 minutes.
3. Add 1-2μL of miniprepped DNA into the thawed electrocompetent cells.
4. Add cell DNA mixture to the chilled cuvette.
5. Dry the outside of the cuvette with a Kimwipe and place in the electroporation apparatus.
6. Pulse the sample using the following settings:
   • Voltage(V) = 2500
   • Capacitance(μF) = 25
   • Resistance(Ω) = 200
   • Cuvette(mm) = 1
7. Recover with 1mL of LB media at 30℃ for 2 hours.
8. Plate 100μL on LB plates with selective antibiotic overnight at 30℃. (a 1/1000 dilution for plating worked well for us)

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7. Acetosyringone Induction Assay

Materials:

• 96 well plate
• LB-Kanamycin media
• 1mL 100mM Stock solution of Acetosyringone (CAS: 2478-38-8) in DMSO
• Overnight cultures of Agrobacterium tumefaciens

Protocol:

1. Inoculate 3 technical replicates per colony of Agrobacterium containing virB1-promoter fusions in the pCAMBIA backbone in 5 mL selective media. Grow at 37°C overnight.
2. Dilute each sample to an OD₆₀₀ of 0.01-0.10
3. Dilute Acetosyringone to a working concentration of 100x in DMSO
4. Plate 200uL samples of each sample into the wells of a 96-well plate with the following treatments
• No dose
• 1μL Acetosyringone (50μM)
• 2μL Acetosyringone (100μM)
5. Measure the optical density (λ = 600) and RFP fluorescence (λ = 580) at t = 0, 1, 2, 3, 4, 5, 6, 7, 8, 18, 24 hours

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8. Plasmid Stability Assay

Materials:

• Autoclaved glass test tubes
• LB media
• LB plates
• LB-Kan plates
• Cuvettes
• Culture ofAgrobacterium tumefaciens carrying pCambia plasmid with ietAS
• Culture ofAgrobacterium tumefaciens carrying pCambia plasmid without ietAS

Protocol:

1. Innoculate 3 biological replicates of Agrobacterium tumefaciens carrying pCambia plasmid with ietAS and Agrobacterium tumefaciens carrying pCambia plasmid without ietAS in 3mL of LB media making a total of 6 cultures.
2. Incubate cultures shaking overnight at 30℃.
3. Take OD600 of each culture.
4. Make 5 dilutions of each culture depending on the OD.
5. Spot plate 5μL each dilution on a LB plate and a LB-Kan plate and incubate for 2 days at 30℃.
6. Record colony count after 2 days.
7. Re-passage the cultures every day in by inoculating 3μL of old culture in 3mL of fresh LB and repeat the previous steps until significant plasmid loss is observed.