电穿孔转化 E.col

This procedure prepares glycerol stock cultures of bacteria for electroporative transformation.

Preparation of Electrocompetent Cells

Materials

•LB Broth Base [Life Technologies, cat # 12795-027]: add 25.0 g per liter of dH2O; autoclave at 121 ℃ for at least 15 minutes. Store at 4 ℃.

•10% Glycerol: autoclave at 121℃ for at least 15 minutes, store at 4 ℃.

•Bacterium to be grown (plate, culture, or stock)

•37℃ Shaker/incubator

•25 mL LB in 125 mL flask (autoclaved)

•250 mL LB in 1000 mL flask (autoclaved)

Procedure

1.Add colony of desired bacterium to 25 mL LB flask; grow overnight.

2.Add 1 mL from overnight culture to 250 mL LB flask; grow until bacteria are in log phase (4-5 hours for E. coli)

3.Split culture across two centrifuge flasks

4.Centrifuge at 2600g for 15 min in a 4 ℃ superspeed centrifuge

5.In cold room or on ice, discard supernatant and resuspend cell pellets in 2 x 125 mL 10% glycerol. FROM HERE ON, ALWAYS KEEP CELLS COLD!!!

6.Centrifuge as before; discard supernatant and resuspend as before; recentrifuge and discard supernatant

7.In cold room or on ice, suspend pelleted cells in 2 x 1 mL 10% glycerol; combine fractions and split into 100 µL aliquots

8.If you are not going to use them immediately, quick-freeze in liquid nitrogen; store at -80 ℃

Electroporation

Materials

•Life-Technologies Cell Porator

•Ice

•1 mL/sample of rich media such as SOC for your cells ro recover in.

•Electroporation chambers (cuvettes)

•Growth plates with proper antibiotic for plasmid.

•Plating rod

•37 ℃ shaking incubator and regular incubator.

Procedure

1.Be sure power is off on the voltage booster and pulse control apparati. If you are not carefull, serious injury could result.

2.Fill the chamber safe with an ice-water slurry. Place the chamber rack in the chamber safe.

3.Label electroparation chambers (cuvettes) to be used, and label microfuge tubes for after electroporation.

4.Place 1-2 µL of your plasmid in the bottom of a labelled eppindorf tube. Add 20 µL of just-thawed electrocompetant cells. Mix.

5.Open electroporation chambers and pipet 20 µL of bacteria-plasmid mixture, suspended between the bosses (electrodes) of the chamber.

6.Handling the chambers carefully, place leaded chamber in a slot in the chamber rack, noting its position. Repeat for each sample. ALWAYS FILL ALL 4 SLOTS WITH CHAMBERS, EVEN IF YOU HAVE LESS THAN 4 SAMPLES TO BE ELECTROPORATED. USE THE DUMMY CHAMBERS LOCATED NEXT TO THE INSTRUMENT OR USE EMPTY CHAMBERS.

7.Once 4 chambers are in place, close the safety interlock lid of the chambersafe and secure.

8.Plug pulse cable into chamber safe.

9.Turn chamber selection knob to your first sample to direct the electrical impulse to the desired chamber.

10.With power still OFF, check settings or set to:

Pulse control = 330 µF

Low Omega

FAST charge rate

11.For E. coli , set resistance to 4kOmega.

12.Turn power switches on for both the power booster and pulse control apparati. Voltage booster will display -16.66.

13.Charge pulse control by setting the CHARGE/ARM switch to CHARGE, then press and hold down the UP voltage control button until the voltage reading is ~410 V. Release button, and quickly switch to ARM setting. The voltage will begin to fall slowly. When it reaches 400 V, press TRIGGER button and hold for 1 second. After pulsing, verify that the pulse control unit indicates <20 V. If you hear a cracking noise, your sample probably had too much salt and has exploded all over the inside of the chamber.