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.