Transformation of XLI-Blue MRF’ Competent Cells

Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States

Transformation of XLI-Blue MRF’ Competent Cells

• Before starting, get water bath at 42°C.
• Add MgCl2 (10 ml/l of 1 M) and MgSO4 (10 ml/l of 1 M) to an aliquot of Super Optimal Broth (SOB).
• Prepare and prewarm SOC (SOB with Catabolite repression) medium to 42°C. Place LB-amp plates at RT.
• Prechill 2059 tubes by placing on ice. Thaw XLI-Blue MRF’ competent cells on ice.
• Gently disperse cells then dispense into bottom of chilled 2059 tubes at 100 µl/tube using plastic 1 ml pipet. Keep on ice.
• Add 1 µl/tube (0.1-50 ng) of plasmid from miniplasmid prep. to cells. Include a no plasmid control. As a positive control, use 1 µl of pUC18
• Incubate on ice for 30 minutes.
• Put tubes in 42°C water bath for exactly 45 seconds.
• Remove and place tubes immediately on ice for 2 minutes.
• Remove from ice and add 0.9 ml/tube of 42°C SOC medium.
• Incubate for 1 hour (37°C) shaking at 225-250 rpm.
• Plate on LB-amp plates using 5, 50, 200 µl/plate. Use glass spreader and rotating platform.
• Incubate overnight at 37 °C.
• Pick colonies using grid plate method (LB-amp plate grid + Miniplasmid Prep.).