- Production of electro
competent E. coli cells
- Electroporation of E.
coli cells
Production of electro
competent E. coli cells
Perform the following steps under sterile conditions
-
Inoculate 5 ml LB and
incubate over night at 37 oC with
150 rpm
Inoculate 2 x 300 ml
preheated LB medium with 1 ml overnight culture
Incubate at 37 oC
with 150 rpm for app 5 hours until OD600
= 0.5
Cool the cultures on ice for 15 minutes
Pellet cells by centrifugation 4500 rpm (Sorvall RC5)
at 4 oC.
Discard the supernatant and resuspend pellet
in 50 ml ice cold sterile MilliQ water
Pellet the cells and discard the supernatant
Wash the cells in 10% glycerol for 1 hour
Pellet the cells and discard the supernatant
Resuspend the cells in 3 ml 10% glycerol
Aliquot the resuspended cells
in 100 ml
portions
and store at
– 80 oC.
Electroporation of E. coli
Aim: To
introduce a vector into E. coli
Materials:
 |
50
µl
electrocompetent cells |
|
500
µl
SOC medium |
|
2 x (LB + selection)
plates |
|
Electroporation cuvette (0.2 mm electrode gap)
|
|
Electroporation apparatus: Bio-Rad Gene Pulser II
(or similar apparatus) |
|
Your vector DNA (typically 1
µl
of Miniprep) |
Procedure
-
Place the electroporation cuvette on ice
-
Get the competent cells from the – 70 oC
freezer
-
Thaw the cells on ice
-
Switch on the electroporation apparatus and change
the settings to the following
Voltage =
2.50 kV (push “set voltage” and then “Raise”)
Capacitance = 25
mF
Resistance = 200
W
-
Place 1
µl
of your DNA (Miniprep) on the internal side to the cuvette
-
Gently use the 50
µl
of competent cells to flush the DNA containing droplet
to the bottom of the cuvette. Gently thump the cuvette to the table a
couple of times to remove air bubbles
-
Dry the exterior sides of the cuvette with a paper
towel (to prevent short circuits)
-
Place the cuvette in the apparatus
-
Push in the two red buttons
simultaneously and hold them in until you hear a beep. (If the
sample explodes then see below*).
-
Remove the cuvette from the apparatus and apply 450
µl
SOC medium as quickly as possible (fast application of SOC increases the
number of surviving cells)
-
Pour the cells into a new 1.5 ml Eppendorf tube
-
Incubate the cells at 37
oC for 1 hour with 350 rpm.
-
Plate the cells onto two selective plates (1/10 and
9/10 of the volume)
-
Incubate the plates at 37
oC over night
-
Clean the used cuvettes: first rinse with 70 %
ethanol, then with water and then 96 %
ethanol. Air dry the cuvette in a UV box
(PCR bench) to degrade any remaining DNA.
* If the salt concentration in the DNA sample is too
high the electric shock will be discharged
very fast resulting in a small explosion, giving a
loud “CLICK" or "BANG”.
Repeat the procedure but with 0.5
µl
of your Miniprep, as the competent cells are killed in
the “explosion”.
|
