OverExpress^™ Electrocompetent Cells

Preparation for Transformation

OverExpress™ Electrocompetent Cells are provided in 25 μL aliquots, sufficient for one transformation reaction. Transformation is carried out in a 0.1 cm gap cuvette. Optimal settings for electroporation are listed in the table below. Note, alternate setting result in 20-50% lower efficiencies. Typical time constants are 3.5 to 4.5 msec.

Electroporation Settings (chart) Optional transformation control reactions include electroporation with 1 uL (10 pg) of supercoiled pUC19 DNA.

Optimal Setting	Alternate Settings	Alternate Settings
1.0 mm cuvette 10 μF 600 Ohms 1800 Volts	1.0 mm cuvette 25 μF 200 Ohms 1400 – 2000 Volts	2.0 mm cuvette 25 μF 750 Ohms 2400 Volts

To ensure successful transformation results, the following precautions must be taken:

• For best results, the ligation reaction must be purified or heat killed at 70 ºC for 15 minutes before transformation.
• The DNA sample to be used for electroporation must be dissolved in water or a buffer with low ionic strength, such as TE. The presence of salt in the electroporation sample leads to arcing at high voltage, resulting in the loss of the cells and DNA.
• Microcentrifuge tubes and electroporation cuvettes must be thoroughly pre-chilled on ice before use.
• The cells must be completely thawed on ice before use.
• Recovery Media must be at Room Temperature prior to use
• For highest transformation efficiency, use the provided Expression Recovery Medium to resuspend the cells after electroporation. Use of TB or other media may result in lower transformation efficiencies and induction of protein expression.

Transformation Protocol

1. Have Expression Recovery Medium and 17 mm x 100 mm sterile culture tubes readily available at room temperature (one tube for each transformation reaction).
   Note: Transformation efficiency may decrease with the use of SOC or other media.
2. Place electroporation cuvettes (1.0 mm gap) on ice.
3. Remove OverExpress cells from the -80 °C freezer and place on wet ice until they thaw completely (10-15 minutes).
4. When cells are thawed, mix them by tapping gently.
5. Add 1 μL of the heat-denatured purified ligation product, to the 25 μL of cells on ice. Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells.
   Note: Failure to heat-inactivate the ligation reaction will prevent transformation.
   Note: Do not use of more than 2 μL of ligation mix may cause electrical arcing during electroporation.
6. Carefully pipet 25 μL of the cell/DNA mixture into a chilled electroporation cuvette without introducing bubbles. Quickly flick the cuvette downward with your wrist to deposit the cells across the bottom of the well. Electroporate according to the conditions recommended in the Electroporation Settings table.
7. Within 10 seconds of the pulse, add 975 μL of Expression Recovery Medium to the cuvette and pipet up and down three times to resuspend the cells. Transfer the mixture to a culture tube.
8. Place the culture tube in a shaking incubator at 250 rpm for 1 hour at 37 °C.
9. Plate up to 100 μL of transformed cells on LB agar plates containing the appropriate antibiotic.
10. Incubate the plates overnight at 37 °C.
11. Transformed clones can be further grown in LB medium.

Sample Induction Protocol

1. Inoculate a single colony from a freshly streaked plate into 5 mL of LB medium containing the appropriate antibiotic for the plasmid and host strain.
2. Incubate with shaking at 37 °C overnight. To minimize the amount of expression of the target protein prior to induction, add glucose to the growth medium at a concentration of 0.2% (w/v).
3. Inoculate 50 mL of LB medium containing the appropriate antibiotic with 0.5 mL of the overnight culture prepared in step 2.
4. Incubate with shaking at 37 °C until the OD600 reaches 0.8 -1.0.
5. Add IPTG to a final concentration of 1 mM. (Prepare a 1 M solution of IPTG by dissolving 2.38 g of IPTG in water and adjust the final volume to 10 mL. Filter sterilize before use). To determine the optimal concentration of IPTG for maximum expression of the target protein, a range of IPTG concentrations from 0.25 - 2 mM should be tested.
6. Incubate with shaking at 37 °C for 3-4 hours. Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.
7. Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10 minutes at 4 °C.
8. Remove the supernatant and store the cell pellet at -20 °C (storage at lower temperature is also acceptable).