Preparation of electrocompetent cells

Overview

We use a protocol commonly used in our host lab provided by Diewertje Piebes. The main advantage of electrocompetent cells compared to chemically competent cells are a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.

Materials

Prechilled detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
Table-top OD600nm spectrophotometer
SOB WITHOUT MgCl
10% glycerol. Sterilise by passing through 0.22-µm filter. Store at 4%deg;C

Procedure

Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80°C TOP10 glycerol stock into and shake overnight at 37°C
Inoculate 200 ml of SOB containing no magnesium with 3 ml of preculture and grow in a 37°C shaker to an OD600 of 0.6-0.8
- please note that E. coli growth rate is significantly reduced due to the absence of Magnesium
- it is possible to add more preculture if you are in a hurry, however, it is not recommended
- Prewarmed medium can shorten preparation time by up to one hour
Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
Note that from this point on the cells, media, bottles and glassware will have to be kept on ice at all times!! If possible it is recommended to work in a cold room.
decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
Once again decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
- This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield
After one final centrifugation step resuspend cells in (2ml???) of ice-cold 10% glycerol
Aliquot 50 μl samples to sterile, prechilled, 1.5 ml or 2 ml eppendorf tubes and flash freeze by immersion in liquid nitrogen
Store at -80°C indefinitely
Test competence (see below)

Measurement of competence

Transform 50 μl of cells with 10 pg of standard pUC19
- This is 1 μl of standard pUC19 Invitrogen plasmid
Hold on ice for 30 minutes
Heat shock 60 sec at 42°C
Add 200 μl SOC
shake at 37°C for 2 hours in 14 ml aerated tubes
Plate 20 μl and 200 on AMP plates using a sterile plate spreader
- Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000
- Competence should be at least 5x10⁷. The higher the better.

Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells

From 2011.igem.org

Contents

Preparation of electrocompetent cells

Overview

Materials

Procedure

Measurement of competence