Stanford/BIOE44:Module 1:Day3

M1: Day 3 - Electrocompetent Cell Prep and Transformation

Introduction

In Class

Electrocompetent Cell Prep

The first two steps of the protocol have been done for you.

1. Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
2. Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60.
3. Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
4. Wash the cell pellet with 20 ml of ice-cold H2O then centrifuge again as above.
5. Resuspend the pellet in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 30 seconds at 4C.
6. Wash the cells again with 1 ml of ice cold H2O and centrifuge as above.
7. Repeat the above wash and spin step.
8. Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.
9. Mix 0.5uL of your ligation reaction with 50μl of electrocompetent cells transfer into a 0.1-cm cuvette.
10. Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance).
11. After electroporation, immediately add 1 ml of SOC and transfer to 1.5mL microcentrigure tube.
12. Incubate cells (with shaking or rotation) at 37C for at least 30minutes
13. Spin down cells for 2 min at 4000g in a microcentrifuge.
14. Resuspend the pellets in 200 μl of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow for 12-16 hours (overnight) at 37C.