Competent E. coli (Rubidium Chloride Method)

Contents

1. Materials
2. Protocol
   • 2.1 Growing the cells
   • 2.2 Making the cells competent
   • 2.3 Aliquoting and storage
3. Notes
4. References

1. Materials

1.1 TFB1 Buffer

• 100 mM Rubidium Chloride
• 50 mM Manganese Chloride
• 30 mM Potassium Acetate
• 10 mM Calcium Chloride
• 15% Glycerol

Adjust pH to 5.8 with acetic acid. DO NOT OVERSHOOT. Manganese will crash out if you attempt to readjust with NaOH. Filter.

1.2 TFB2 Buffer

• 10 mM MOPS or PIPES
• 10 mM Rubidium Chloride
• 75 mM Calcium Chloride
• 15% Glycerol

Adjust pH to 6.5 with KOH. Filter.

Store buffers at room temperature or 4 C. Use while ice cold.

2. Protocol

2.1 Growing the cells

1. Streak desired commercial cells on an LB plate and grow at 37C for 24 hours.
2. Choose a single colony and grow in 5 mL of LB overnight at 37C and 230 rpm.
3. Prepare 250 mL LB + 20mM MgSO4 in a 2L baffled flask and inoculate with 3 mL of overnight culture.
   1. Shake at 37 C and 250 rpm until OD= 0.4-0.6.
4. Transfer cells to 5 50 mL conicals and centrifuge to pellet.
   1. Pre-chill the conicals on ice and bring the centrifuge down to 4 C prior to transferring cells. Swing bucket rotor, 4500 x g, 5 minutes.

2.2 Making the cells competent

From here on, all steps are completed in the cold room on ice.

1. Discard supernatant and resuspend each pellet with 20mL of TFB1, gently pipetting to solubilize.
   1. Be patient and gentle. Do not vortex cells or pipette too violently.
2. Combine the pellets in 2 50 mL conicals and incubate on ice for 5 minutes.
3. Pellet cells; swing bucket rotor, 4500 x g, 5 minutes, 4 C.
4. Resuspend each pellet in 5 mL TFB2, pipetting up and down gently to mix.
5. Combine cells into one conical and incubate on ice for 45-60 minutes.

2.3 Aliquoting and storage

1. Aliquot 50uL into pre-chilled 0.6mL tubes.
2. Flash freeze with liquid nitrogen.
3. Store at -80 C and update the racks database to reflect the inventory.

3. Notes

E. coli cells can be very sensitive to temperature and perturbation, so be careful and keep everything as cold as possible.

Competency can be verified by transforming a plasmid and plating with the appropriate antibiotic. Using this protocol, I have been able to transform up to 4 purified plasmids with different antibiotics successfully.

4. References

Protocol adapted from: https://www.med.unc.edu/pharm/sondeklab/files/resource-files/competent-cell-dna-protocols/Competent_cell_RbCl.doc.