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1. Have Recovery Medium and 17 mm x 100 mm sterile culture tubes readily available at room temperature (one tube for each transformation reaction). Transformation efficiency may decrease with the use of SOC or other media.

2. Place electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microfuge tube for each transformation reaction).

3. Remove Electrocompetent Cells from the -80 °C freezer and place on wet ice until they thaw completely (10-15 minutes).

4. When the cells are thawed, mix them by tapping gently. Aliquot 25 μL of cells into the chilled microcentrifuge tubes on ice.

5. If using ligation buffer from any Lucigen cloning or ligation kit, add 1 μL of the heat-denatured ligation reaction to the 25 μL of cells on ice. Failure to heat-inactivate the ligation reaction will prevent transformation. Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells. Use of more than 2 μL of ligation mix may cause electrical arcing during electroporation. For ligation reactions using other commercial kits, please refer to the manufacturer’s instructions

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 above.

7. Within 10 seconds of the pulse, add 975 μL of Recovery Medium to the cuvette and pipet up and down three times to resuspend the cells. Transfer the cells and Recovery Medium to a culture tube.

8. Place the tube in a shaking incubator at 250 rpm for 1 hour at 37 °C.

9. Spread up to 100 μL of transformed cells on LB (or other nutrient media) agar plates containing the appropriate antibiotic.

10. Incubate the plates overnight at 37 °C.

11. Transformed clones can be further grown in TB or in any other rich culture medium.

Reference: https://www.lucigen.com/docs/manuals/MA053-Phage-Display.pdf

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