Preparation Of Competent Cells For Electroporation

1. Carry out step 1 through step 4 in the preceding section.

2. Extensively wash the cells with 100 ml distilled water or low-salt buffer in order to reduce the ionic strength of the cell suspension.

3. Centrifuge at 5000 x g for 10 min at 4°C and carefully decant the supernatant.

4. Repeat step 2 and step 3 twice.

5. Resuspend the cells in 20 ml of low-salt buffer or distilled water. Add glycerol dropwise with gentle swirling to 10% (v/v). Aliquot the cells at 0.2 ml/tube, freeze and store at -80°C until use.

Reagents Needed

LB (Luria-Bertaini) Medium (per Liter)

10 g Bacto-tryptone

5 g Bacto-yeast extract

5 g NaCl

Adjust to pH 7.5 with 0.2 N NaOH and autoclave.

LB Plates

Add 15 g of Bacto-agar to 1 l of freshly prepared LB medium and autoclave. Allow the mixture to cool to about 55°C and add the appropriate amount of antibiotic stock. Pour 25 to 30 ml of the mixture into each 100-mm Petri dish in a sterile laminar flow hood with filtered air flowing. Remove any bubbles with a pipette tip and let the plates cool for 5 min prior to covering them. Allow the agar to harden for 2 h and partially dry at room temperature for a couple of days. Store the plates at room temperature for up to 10 days or at 4°C in a bag for 1 month. Allow the cold plates to warm up at room temperature for 1 to 2 h prior to use.

Protocol 2. Transformation of Cells by CaCl2 Method

1. Thaw an aliquot of 0.2 ml of frozen CaCl2-treated competent cells on ice.

2. Assembly transformation as follows:


Components 12 3

Cells 50 ml 50 ml 50 ml

Tip: 15 to 20 ml of ligated mixture can be directly added into 50 to 60 ml of competent cells. 3. Incubate on ice for 50 to 60 min.

4. Heat shock at 42°C for 1 to 1.5 min and quickly place the tubes on ice for 1 min.

Tip: Heat-shock and chilling on ice should not extend past the time indicated.

Otherwise, the efficiency of transformation will decrease 10- to 20-fold.

5. Transfer the cell suspension to sterile culture tubes containing 1 ml of LB medium without antibiotics. Incubate at 37°C for 1 to 2 h with shaking at 140 rpm to recover the cells.

6. Transfer 50 to 150 ml of the cultured cells per plate onto the center of the LB plates containing 50 mg/ml ampicillin (or 0.5 mM IPTG and 40 mg/ml X-Gal for color selection). Immediately spread the cells over the entire surface of the LB plates using a sterile, bent glass rod.

7. Invert the plates and incubate them at 37°C overnight. Selected colonies should be visible 14 h later.

Protocol 3. Transformation by Electroporation

1. Thaw an aliquot of 200 ml of frozen, non-CaCl2-treated competent cells on ice.

2. Chill three disposable microelectroporation chambers (BRL) on ice.

3. Connect the power cable to a BRL-Porator pulse control + power supply apparatus, to BRL-Porator voltage booster, and between these two units.

4. Set a pulse control as follows:

Power Charge

Capacitance (mF) 330

High ohm/low ohm Low ohm

Charge rate Fast

Set up the voltage booster at 4 kV for E. coli

5. Add ice water to the chamber safe up to 4/5 of the volume, and place the chamber rack in the chamber safe.

6. Thaw three aliquots (A, B, C) of 20 ml of competent cells on ice. To aliquot A, add 3 ml of DNA solution containing 1 mg DNA. To aliquot B, add 3 ml of DNA solution containing 2 mg DNA. To aliquot C, add 3 ml of DNA solution containing 4 mg DNA. Gently mix and place on ice until use.

Note: Try to avoid any air bubbles during mixing. The total volume of the transformation mixture should be <25 ml.

7. Use a pipette tip to transfer one aliquot and carefully place the mixture drop between the electrode poles in the microelectroporation chamber. Carefully cover the chamber.

Note: The liquid should not drop to the bottom of the chamber.

8. Gently place the chamber into the cell in the safe rack, cover the chamber and turn the electric-shock pointer toward the cell containing the transformation chamber.

9. Connect the power from the voltage booster to the chamber safe, and turn on power for the pulse control and voltage booster.

10. Press charge button on the pulse control up to 365. When the DC voltage drops down to 345 to 350 V, turn the button from charge to arm. Quickly push the trigger button for 1 to 2 s. The voltage goes down to <10 and the kV number in the voltage booster should be 1.9 to 2.0.

11. Turn off the power and gently remove the chamber from the cell. Note: The transformed cell suspension should still be between the positive and negative electrode poles. It is not a good sign if the liquid drops to the bottom.

12. Quickly transfer the transformed cell suspension into a test tube containing 0.5 ml LB medium without ampicillin because the cells are quite weak. Mix well and place at room temperature for not longer than 15 min.

13. Repeat the preceding steps until all the samples are transformed.

14. Incubate at 37°C for 1 to 2 h with shaking at 150 rpm to recover the cells.

15. Use a sterile, bent glass rod to spread 20, 50, 100, and 200 ml of each of the three transformant cells over the entire surface of LB plates containing 50 mg/ml ampicillin.

16. Invert all the plates prepared and incubate at 37°C for 12 to 16 h until colonies are visible.

0 0

Post a comment