- Overnight E. coli culture
- 50 mL Falcon tubes
- 1.5 mL centrifuge tubes
- Freezer box
Dilute an overnight culture of E. coli 1:100 in LB media. Grow to an OD of 0.3 and then put the culture on ice. Transfer the culture to chilled 50 mL Falcon tubes and centrifuge at 5,000 g for 10 minutes at 5°C. Discard the supernatant and resuspend at 1/10th volume of cold TSS by gently pipetting up and down. Transfer 100 μL aliquots to chilled 1.5 mL centrifuge tubes, freeze in liquid nitrogen, and then store in the -80 freezer.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- This protocol is adapted from Chung and Miller (1989). One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci., 86(April), 2172–2175.
- When making TSS, first add PEG to LB, heat in a 50°C water bath, and then swirl to mix. Add Mg2+, wait until the solution gets to room temperature before adding DMSO, and filter sterilize. Store in the 5 degree fridge.