Preparing e. coli competent cells

Updated 6/23/2014 02:10 AM

by Johan Sosa

Introduction

Preparing competent cells from e. coli. This protocol is adapted from notes provided to me by Mithu B. I have tested this with K₁₂ strains such as dh5a, MG1655, and HB₁₀₁.

Materials

Culture of e. coli cells to be made competent.

1 LB Agar plate

250mL Erlenmyer flask

53mL LB liquid broth

1 bacteria culture tube

50mL sterile centrifuge tubes (upto 5)

3mL chilled CaCl₂

Procedure

Day 1

Streak e. coli cells onto LB plate in a manner that creates single colonies.

Grow overnight at 37°C.

Day 2

Select a single colony from the LB plate and use it to inoculate 3mL of LB broth in a bacteria culture tube.

Grow overnight in a shaker at 37°C.

Day 3

Add to 50mL of LB broth (1:100 dilution) in a sterile 250mL Erlenmyer flask.

Use a pipette to add 50µL of the cells from the 3mL growth to the 50mL LB broth in the Erlenmeyer flask.

Agitate at room temp for 12 - 16 hours. This will put the cells into the log growth phase. If waiting longer then use a higher dilution like 1:200. For faster results: -> Pour 3mL of the overnight culture into 10-20mL of LB in a flask and agitate for 2.5-3 hours at 37°C.

Pour 10mL aliquots of cells in log growth phase into individual 50mL centrifuge tubes.

Place centrifuge tubes in centrifuge and spin at 3000 rpm/4°C for 10 minutes.

Pour off the supernatant and suspend pellet in 2 - 3mL of chilled CaCl₂ (20 - 75 mMolar). Immediately store in -80°C freezer or in dry ice.