Cong T. Trinh:Electroporation

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Electroporation method 1 using 2mm cuvette

Electrocompotent cells

  • Grow 10mL of the target cells in LB in a 14mL culture tube to an O.D of 0.5 to 0.7.
  • Chill the culture on ice.
  • Once the cell culture has chilled (~10 min), transfer the culture into chilled 15mL centrifuge tubes.
  • Centrifuge the tube and discard the supernatant.
Centrifuge at 4°C at 4,000 RPM for 10 minutes.
  • Wash the chilled culture with 5mL of chilled water three time, each time discarding the supernatant.
Centrifuge at 4°C at 4,000 RPM for 10 minutes.

Resuspend the cells in 250μL of sterile water. Distribute the cells into 5, 1.5mL tubes (50μL per tube)

One electroporation reaction uses 50μL. Store the remaining 4 tube in the stock box in the -80°C freezer.

Preperations before starting

  • Place the electroporation cuvette on ice (keep sterile packaging unopened)
  • Warm plates with the selected antibiotic to 37°C
  • Prepare SOC media (1mL per reaction)

Setting the Electro cell manipulator

  • Set voltage to 2.5kV
3.0kV may increase efficiency in some situations
  • Time should be ~5 msec

Procedure

  • After the washing steps to prepare the electrocompetent cells resuspend the cell pelette in 50μL of chilled water and transfer to a sterile 1.5 mL eppendorf tube.
  • Add 2μL of ligated DNA (the Plasmid to be inserted). Swirl gently with the pipette tip.
  • Place the tube back on ice for ~1 minute to chill.
  • Transfer the contents of the tube to a 2mm electroporation tube, gently tap the cuvette to bring all of the solution to the bottom of the cuvette.
  • Quickly wipe the electroporation tube of any condensates and insert the electroporation tube into the electro cell manipulator. Press the pulse button and observe the time (should be ~5 msec.)
  • Quickly add 1mL of warmed SOC and place the electroporation tube into a 37°C incubator for 30-60 minutes.
  • After an hour, transfer the content of the electroporation cuvette to a 1.5mL tube and centrifuge for 5 minutes at room temperature. (optional) Resuspend in 50μL of sterile water.
In order to get all of the cells from the electroporation cuvette, you can place a 10μL tip onto the end of a 1000μL to reach the bottom of the cuvette.
  • Plate the prewarmed plates with the correct antibiotic and incubate at 37°C overnight.

Electroporation alternative method using 1mm electroporation cuvette

Step 1. Pick a colony from a plate (or use 100 uL of overnight culture) of the knockout strain containing the kanamycin cassette and transfer it into a sterile baffled shake flask containing 20 mL of LB. Grow cells from 6-8 hours. When OD600nm reaches between 0.5 and 1, proceed to Step 2.

Step 2a. Place the baffled shake flask on ice for 15-30 min. Gently swirl the flask a couple of times while waiting to help cooling.

Step 2b. Transfer the cell culture to a 50 mL pre-chilled tube and centrifuge it at 5000 rpm for 10 min. (Use 5uL of cells each and spread it on the LB/30kan and LB/100 Amp plates).

Step 2c. Wash the cell pellet with the cold sterile water and centrifuge the re-suspended cells at 5000 rpm for 10 mins.

Step 2d. Repeat step 2c once.

Step 2e. Remove all supernatant. Re-suspend cell pellet in 50 uL of the cold sterile water.

Step 3a. Transfer 25 uL of cells into the pre-chilled 1.5 mL tube.

Step 3b. Add 1 μL of the plasmid pCP20 (>20 ng/uL) into the 1.5 mL tube (avoid introducing small bubbles),

Step 3c. Transfer the cells and plasmids from the 1.5 mL tube into the electroporation cuvette. Make sure to clean the metal wall to avoid the electric arcing.

Step 3d. Electroporate cells at the following set parameters - 1800 V, 25F, 200 Ohm.

Step 4a. Transfer the electroporated cells from the cuvette into a tube containing 500uL of pre-warmed SOC medium.

Step 4b. Incubate the tube in the 30oC shaker for 1 hr. (Note that pCP20 is the temperature-sensitive plasmid)

Step 5. Spread 50uL of cells on the LB/100Amp plate and incubate it in the 30oC incubator.


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