BioProtocols--Molecular Cloning

Molecular Cloning

E. coli Transformation for Subcloning

1. Thaw 50 uL frozen competent cells (AG1, Stratagene #200232) per transformation on ice.

2. Transfer the cells to a pre-chilled Falcon 2059 (round-bottom with cap) tube, and add
0.85 uL 2-mercaptoethanol which is provided with the competent cells.

3. Swirl the tube gently for every 2 min while incubating the cells on ice for 10 min.

4. Add an appropriate amount of DNA (1-50 ng) to the competent cells, and incubate on ice
for 30 min without disturbing.

5. Heat shock for 45 sec at 42 C and incubate on ice for 2 min.

6. Add 450 uL pre-warmed (42 C) SOC medium (GibcoBRL), and incubate at 37 C with
vigorous shaking.

7. Plate the cells on 3 same plates (e.g. 50, 150, 300 uL) with glass spreader.

8. Incubate overnight at 37 C.