Latest revision as of 13:36, 3 September 2015

Escherichia coli DH5α Electroporation transformation protocol

In this protocol we make electrocompetent cells and then transform them. This protocol is based on the Eppendorf application note for Escherichia coli DH5α.

Most of this protocol is supposed to be done between 2 and 4C, so its important to prepare the items and move quite quickly through the steps.

Escherichia coli DH5α Electroporation transformation protocol Prepare overnight cultures (for use ~8 hours later) Put required items on ice Prepare petri dishes Prepare 3 LB and 3 LBA plates Making electrocompetent cells:

Host Escherichia coli DH5α injected DNA pUC 19 Growth Medium LB Washing Solution Ice-cold water Recovery Medium SOC (or LB+glucose)

Prepare overnight cultures (for use ~8 hours later)

Step 1) Take 2 new/sterile 50ml falcon tubes and add to each 10ml of sterile LB broth.

(there is usually an autoclaved 1 L duran flask of LB in the fridge, use 20ml from that and transfer to tubes under the flow hood)

Step 2) inoculate the 2 falcon tubes from DH5α stock using a flamed loop.

Step 3) Incubate at 37 °C with shaking overnight, or for 8 hours

Step 4) It should look something like this

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Put required items on ice

The cells need to be kept cold during the procedure. Therefore the cuvettes and falcon tubes should be placed in the cold store of the freezer before starting;

It is also useful to have ice cold RO water, chill before starting

� Prepare petri dishes

Prepare 3 LB and 3 LBA plates

Making electrocompetent cells:

Step 1) Setup an ice bath that will hold several 250ml flasks and the RO water bottle. You will need to use it regularly. Remember to return the water bottle to the ice bath between steps

Step 2) Autoclave 2 x 250ml Duran flasks each containing 90 ml LB medium (leave the lids loose during autoclaving, as trapped gas might cause an autoclave explosion)

Allow the flasks to cool to room temperature (or cool under running water)

Step 3) Inoculate the flasks by pouring in the 10 ml of the overnight culture from the falcon tube into each to make up to 100ml.

Step 4) Place in the incubator at 37 °C with shaking

Grow to an OD600 of approximately 0.5. The culture turbidity should look roughly like this

After removing from the incubator, chill both flasks in an ice bath for 15 minutes

Transfer from the 2 flasks into prechilled falcon tubes.

When done the 2 flasks should fill 4 falcon tubes completely.

Harvest by 20 minutes at 5000 rpm in the JOUAN centrifuge. A pellet should form in the bottom of the tube.

Pour off the supernatant from each tube, taking care not to dislodge the pellet.

Resuspend the pellet by adding 5 ml ice-cold water to each falcon tube and pipette mixing.

Mix the 4 x 5ml resuspended cultures into a single falcon tube. (This saves repeating all the steps for each tube)

Wash the cells by pelleting and resuspending 3 times:

3 times: Pelleting by 12 minutes centrifugation at 5000 rpm. (Use a counter balancing tube.) Then resuspend the pellet in 45 ml of ice-water

Finally resuspend the cells in ice-cold water to a final concentration of approximately 2 x 10^11 cells/ml. (This is usually just a drop or 2-3ml of liquid)

It looks something like this;

Electroporation of cells:

Step 1) Add 90 µl electrocompetent cells to a prechilled cuvette.

Step 2) Add 10 µl of plasmid to cuvette and mix by pipette mixing

Step 3) plate out a -ve control of e-competent cells. Mark plate “LBA/E-comp”

Step 4) Wipe moisture from the cuvette and insert the cuvette into the device.

Electroporation: Use the following parameters Mode Prokaryotes Voltage (V) 1,700 V Time constant (τ) 5 ms

Step 5 Immediately add 1 ml recovery medium (SOC or LB+glucose) into the cuvette. Pipette mix to ensure cells can access media.

Step 6) Incubate 30- 60 minutes with moderate shaking at 37 °C.

Put the cuvette containing the Cells and the SOC in the incubator for 30-60 mins at 37C

Step 7) Plate on LBA. Grow overnight.

Expected results: Transformation efficiency up to 3 x 109 transformants/µg of DNA.

(THIS IS MISSING A CONTROL PLATE)

References Eppendorf AG ΠApplication Hotline ΠD-22331 Hamburg Phone +49 180 3 666 789 ΠFax +49 40 53990 125 Πe-mail: application-hotline@eppendorf.de Adapted from: High Efficiency Transformation by Electroporation ΠShort Protocols in Molecular Biology Second Edition ΠGreen Publishing Association and John Wiley & Sons, New York Π1-22 Р1-23