Preparation of competent E.

coli cells
Written by Steve Doyle (s.doyle@latrobe.edu.au) - March 2014

Adapted from: Sambrook & Russell, Molecular Cloning 3rd Edn. 1.112-1.115 “Protocol 24:
The Inoue method for preparation and transformation of competent E.coli”

- competent cells are used in molecular biology to amplify plasmid DNA that contains
a DNA sequence of interest
- the term “competent” means that they are able to be transformed or take up
plasmid DNA; to enable this, they need to be prepared by treating them with certain
chemicals
- there are a number of different ways to make competent cells – this is the main
protocol that gets used in our lab. You can also purchase competent cells from a
number of different providers, however, they can be quite expensive, depending on
how often you plan to use them.
- we tend to use a particular E. coli strain – TOP10 – from Life Technologies, but there
are a range of different
- competent cells can be difficult to make well, and can take practice. After a batch is
made, they should be tested to determine how competent they are by transforming
a plasmid of known concentration, and also tested to make sure they are not
resistant to any of the antibiotics that you plan to use
- Relevant reading/links associated with the section
o Sambrook & Russel, Molecular Cloning 3rd Edn. 1.112-1.115 “Protocol 24: The
Inoue method for preparation and transformation of competent E.coli”
 This is an extremely good molecular biology resource if you have not
used it already
o Transformation efficiency:
(http://en.wikipedia.org/wiki/Transformation_efficiency)

Reagents
- DMSO
- Luria-Bertani (LB) broth
o 10 g Tryptone, 5 g Yeast extract, 5 g NaCl
o sterilize by autoclaving
- Inoue Buffer
o Prepare 0.5 M PIPES or HEPES (pH 6.7). Sterilize by filtration. Store either 4°C
or -20°C
o Dissolve in 800 mls milliQ H2O
 10.88 g MnCl2.4H2O (55 mM final con.)
  2.20 g CaCl2.2H2O (15 mM final conc.)
 18.65 g KCl (250 mM final conc.)
 20 mls PIPES or HEPES (10 mM final conc)
 milliQ H2O to 1 liter
o Sterilize by filtration. Aliquot and store at -20°C
- Dry ice or liquid nitrogen

Protocol

Day 1
- Usually, you’ll have a tube or two of the previous batch of competent cells left in the
-80C freezer. It is a good idea to keep one tube in a master stock box to ensure you
do not run out.

1. Make 500 ml of LB broth, and autoclave to sterilize

2. Add 5 ml of LB broth to a 10 ml tube
3. using a sterile pipette tip in a sterile environment, scrape the surface of the frozen
culture with the pipette tip, and inoculate the 5 ml of LB broth
4. place tube into a 37C shaking incubator (~180 RPM) overnight

Day 2
1. Late In the afternoon (5 PM) inoculate 1 L flask containing 250 mls LB with O/N
starter

2. Incubate flasks shaking at room temperature (18-22°C) overnight

3. Check OD600 in the morning using a spectrophotometer. Monitor OD600 until reaches
approximately 0.55.

4. When OD600 is reached transfer culture flask to ice bath for 10 minutes

5. Harvest cells by centrifugation, 2,500g for 10 minutes at 4°C

6. Discard all supernatant

7. resuspend cells very gently in 80 ml ice cold Inoue buffer

a. don’t vortex or pipette up and down, just gently shake

8. Harvest cells by centrifugation, 2,500g for 10 mins at 4°C

9. Discard all supernatant

10. Resuspend cells very gently in 20 ml ice cold Inoue buffer

11. Add 1.5 ml DMSO. Mix gently but thoroughly by swirling, and store on ice for 10
minutes
a. Chill sterile microcentrifuge tubes

12. working quickly, dispense aliquots into chilled sterile microcentrifuge tubes. Put
straight into dry ice or liquid nitrogen to freeze.

13. store at -70°C until needed