Laboratory demonstartion

Title: Preparation of E. coli Competent Cell

Introduction

Foreign DNA can be placed in cells by several methods. If the foreign DNA is introduced
into the cell in a form acceptable to the host, genes on that DNA can be expressed and
the DNA can be propagated by the cells. In many cases this is done by attaching the
foreign DNA to a piece of DNA that is capable of replicating within the host. For bacteria
and some eukaryote species, plasmids or phages represent suitable vectors.

The overall process of changing the phenotype of a bacterium by introducing a plasmid

into it is called transformation. Bacteria may be transformed with plasmids by several
techniques. The simplest is merely incubating the plasmid with bacteria whose cell wall
has been weakened.

In this lab demonstration, a kit (TransformAIDTM Bacterial Transformation Kit) is used to

prepare competent cells within a total time of 50 min (from overnight bacterial culture) or
2.5 hr from bacterial colonies.

Procedures:
1) Add 150 µl of overnight culture to a 1.5 ml of pre-warmed C-medium. Incubate 20
min at 37oC in a shaker.
2) Pellet bacterial cells by 1 min centrifugation, discard the supernatant
3) Resuspend cells in 300 µl of T-solution. Incubate on ice for 5 min
4) Centrifuge for 1 min in a microcentrifuge tube, discard the supernatant
5) Resuspend pellet cells in 120 µl of T-solution. Incubate 5 min on ice
6) Add up to 5 µl of ligation mixture (containing 10-100ng vector DNA) or 1 µl of
supercoiled DNA (10-100 pg) into new microcentrifuge tubes. Chill on ice for 2
min
7) Add 50 µl of the prepared cells to each tube containing DNA, mix and incubate
on ice for 5 min.
8) Plate immediately on pre-warmed LB antibiotic agar plates. Incubate overnight at
37 oC.

Based on this lab demo, the students should prepare a group report of 3 pages. In the
report, include the following points:
1) Introduction to competent cells
2) Why do we need to prepare competent cells?
3) The conventional methods available to prepared competent cells?
4) What are actually C-medium and T solution in this kit?
5) The optimum concentration of E. coli cells used in the preparation of competent
cells
6) Briefly describe one application of this technique that you can think off?
7) How has this session benefitted you?

Reference:
1) Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli
Using Salt-free Growth Medium", Biotechniques 20, 42-44 (1996).