Heat Shock Transformation with DH5Cells
1. Remove competent cells from -80ºC freezer and thaw on wet ice. Place required number of
sterile 1.5 mL microcentrifuge tubes on wet ice.
2. Gently mix cells, then aliquot 100 l competent cells into chilled microcentrifuge tubes.
3. To determine transformation efficiency, add 5 l (0.5 ng) control pUC19 to one tube
containing 50 l competent cells. Move the pipette through the cells while dispensing.
Gently tap tube to mix.
4. Add 1-3 l (1-10 ng of DNA) of the DNA ligation reaction directly to a second tube
containing 100 l competent cells, moving the pipette through the cells while dispensing.
Gently tap tube to mix.
5. Incubate cells on ice for 30 minutes.
6. Heat-shock cells for 45 seconds, at 42ºC. Do not shake.
7. Place on ice for 2 minutes.
8. Add 0.95 mL of room temperature SOC medium
9. Shake at 225 rpm for 1 hour at 37ºC for expression
Updated 11/23/09 by RK