IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation protocol 3

Rudner Lab

Reagents

10xMC: (10ml) 1M Potassium Phosphate pH7.0 (1.36g) 30mM sodium citrate (0.088g) 20% Glucose (2g) 220mg/ml Ferric Ammonium Citrate (2200mg) 1% Casein Hydrolysate (0.1g) 2% Potassium Glutamate (0.2g)

1xMC + 3mM MgSO4: 900 μl ddH2O 100 μl 10xMC (stored at –20oC) 3 μl 1M MgSO4 (100ml stock 1M MgSO4 - 24.627g)

LB plate PstI and EcoRI Plasmid DNA (concentration not specified) Plate containing DH5α or XL1-Blue

Protocol

Day 1

• Inoculate 3x1ml 1xMC (+3mM MgSO4) from a plate of B.subtilis in a 10ml tube and place in the shaking incubator at 37oC for 4 hours
• Shortly before 4 hrs have elapsed prepare 5ml tubes for transformation by adding

9μl and 1μl of this solution used to transform. At the end we want 2x19μl volumes and 2x 1μl volumes and one empty control tube.

• After 4 hrs of growth add 20-200μl of competent cells to the transformation tubes
• Place transformation tubes back in the incubator for 37^oC and shake for 2 hours
• Plate the entire transformation on selective medium corresponding to the antibiotic resistance of the plasmid/construct. If too many transformants are yielded then use 1/20th (~50μl) of the transformation on one plate and the rest (~950μl) on a second plate.
• Incubate overnight at 37oC