RbCl comp cells preparation

This rubidium chloride protocol gives better transformation efficiencies than the CaCl2 procedure for most strains. It's an adaption of one described in the QIAexpressionist.

Materials

• LB medium and plates with appropriate antibiotics
• TFB1, ice-cold
• TFB2, ice-cold
• dry ice / isopropanol bath

TFB1

• 30 mM potassium acetate
• 10 mM CaCl2
• 50 mM MnCl2
• 100 mM RbCl
• 15% glycerol Adjust pH to 5.8 with 1 M acetic acid. Filter-sterilize (0.45 µM) and store at RT.

TFB2

• 10 mM MOPS or PIPES (pH 6.5)
• 75 mM CaCl2
• 10 mM RbCl
• 15% glycerol Adjust pH to 6.5 with 1 M KOH. Filter-sterilize (0.45 µM) and store at RT.

Protocol

1. Inoculate a single colony from an LB plate into 2.5 ml of LB medium + antibiotics (if necessary for your cells). Incubate overnight at 37 °C with shaking at 225 rpm.

2. Subculture the overnight culture 1:100 by inoculating 1 ml into 100 ml of pre-warmed LB + antibiotics (if necessary). Grow the cells in a 250 ml flask until the OD600 reaches 0.4-0.6. Timing depends on the strain.

3. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.

4. Pellet the cells by centrifugation at low speed (4000 xg for 5 minutes at 4 °C) and discard the supernatant. Always keep the cells on ice.

5. Gently resuspend the cell pellet in ice-cold TFB1 (30 ml for a 100 ml culture). Combine the resuspended cells in one bottle. For the remaining steps, keep the cells on ice and chill all pipettes, tubes, and flasks.

6. Incubate the resuspended cells on ice for 90 minutes at 4 °C.

7. Pellet the cells by centrifugation at 4000 xg for 5 minutes at 4 °C. Discard the supernatant carefully and keep the cells on ice.

8. Gently resuspend the cells in 4 ml of ice-cold TFB2.

9. Incubate the cells on ice for 15-60 minutes, then aliquot 100 µl/tube for storage at -80 °C. Quick-freeze the tubes in a dry ice/isopropanol bath or in LN2.