"

From 2012.igem.org

Contents 1 Preparation of electrocompetent cells of E.coli 1.1 Reagents and Materials 1.2 Procedure 1.2.1 Day 1: 1.2.2 Day 2: 1.2.3 Day 3:

Preparation of electrocompetent cells of E.coli

Reagents and Materials

Procedure

Day 1:

• Inoculate appropriate strain from -80 degree Celcius stock to a LB agar plate and incubate at 37 degree Celcius o/n.

Day 2:

• Inoculate 10 ml SOB medium with a single colony from the plate and incubate o/n shaking at 37 degree Celcius.

Day 3:

preparations in advance (can be made while cells are growing:

1. Take two or three big buckets of ice and add a little water. Leave them at RT for a while so that a layer of ice-cold water will form at the bottom.
2. Put 50 empty 0.5 ml tubes (closed caps) in a box and store them at -80 degree Celcius.
3. Place your glycerol, MQ and empty Beckmann-tubes on melting ice in the cold room. Make sure you have bucket of melting ice ready to chill your cells in. Furthermore, make sure you have in the cold room:
   • Yellow and blue pipette tips and the yellow blue pipettes
   • one disposable 10 ml pipette
   • an automatic pipette
   • the SLA 3000 rotor
   • scales

Half an hour before your cells are at their desired OD (approximately 2.5 hours after inoculation), switch on the Beckmann-centrifuge and pre-chill it at 4 degree Celcius.