Bacillus Genetic Stock Center Catalog of Strains Seventh Edition ...

Electroporation ProtocolAdapted from:Macaluso and Mettus. 1991. J Bacteriol 173:1353-1356.Materials:culture of B. thuringiensis host on solid mediumplasmid DNA, prepared from B. megaterium or E. coli dcm - host100 ml stock of BHIG (Brain Heart Infusion + 0.5% glycerol), sterile10 ml of BHIG in a 250 ml flask, sterile95 ml of BHIG in a 1 or 2 liter flask, sterile150 ml of EB (0.625 M sucrose, 1 mM MgCl 2 ), sterile and chilled on icetwo sterile centrifuge bottlessterile cryovials for storage of electro-competent cells, if desired0.4 mM electroporation cuvettes (BioRad)BioRad GenePulser ® with Pulse Controller ®sterile test tubesselective agar plates (LB agar plus appropriate antibiotic)Protocol:Day 11. Inoculate 10 ml of BHIG in a 250 ml flask with a single Bt colony.2. Incubate at 30°C with moderate shaking overnight.Day 2Cell Preparation1. Dilute 5 ml of overnight culture into 95 ml BHIG in 1 or 2 liter flask.2. Incubate 1 hr at 30°C with vigorous shaking.3. Pellet cells by centrifugation (at an appropriate speed for your machine)4. Suspend in 100 ml cold EB. Note: Be careful to keep cells cold from this point forward untilthe electric pulse has been delivered during transformation.5. Repeat centrifugation.6. Suspend in 50 ml cold EB.7. Store 2 ml aliquots of the cell suspension in cryovials and store in -70°C for later use, ifdesired.Transformation1. If using frozen cells, thaw them on ice. Transfer 0.8 ml aliquot of cell suspension to a 0.4 cmcuvette; mix in up to 10 µl DNA.2. Chill on ice 5 minutes.3. Apply a single pulse at a setting of 2.5 kV, 25 µF, 5 Ω.4. Dilute cell suspension with 1.6 ml BHIG.5. Incubate at 30°C with moderate shaking for 1 hr.6. Plate dilutions on selective agar plate and incubate overnight.29/Back to Table of Contents