Team:Groningen/2-step Transformation Procedure for Bacillus subtilis 168

From 2010.igem.org

2-step transformation procedure for Bacillus subtilis 168

• Cells are grown O/N at 37 °C in supplemented medium contains Spizizen's salts (without antibiotics)
• Dilute cells 10X in fresh medium (10 ml) and grow for 3 h at 37 °C, shaking
• Make agar plates
• Cells are then diluted 1:1 in starvation medium(prewarmed, no antibiotic)
• After 2 hours, approx. 1 μg DNA (BRB689; Cmr amyQ+) is added to 100 μl cells in a 2 ml tube, and the mixture is incubated for 30 min at 37 °C (200 rpm)
• Dilute 3-fold in prewarmed TY medium and plate after another 45 min (37 °C;200 rpm)
• Dilutions (in starvation medium) for plating are:

* 10^0 and 10^-1 on selective media
* 10^-5 on non-selective plates (viable count)