Note: This is a protocol for C2987P.
Transformation Protocol Variables
- Chill a metal 96-well block on ice.
- Remove the plate from -80°C freezer, and place in chilled metal 96-
well block (or directly on ice) for 2 minutes to thaw the competent
cells. (Alternatively, a 24-well segment could be separated from the
plate by placing the 96-well plate on dry ice, bending the plate at
the connections, and cutting the foil seal between the sections. The
unused sections could be returned to -80°C freezer.)
- Carefully remove the aluminum foil seal from the plate or pierce holes
through the foil seal with pipette tips.
- Add 1-2 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture
using a multichannel pipette. Carefully swirl the tips to mix cells and
- Seal the plate with plate cover, or cap strips, or tapes.
- Incubate the plate in the chilled metal block (or on ice) for 20 minutes.
- Heat shock the cells at exactly 42°C for exactly 10 seconds by
transferring the plate to a pre-warmed thermal block or water bath.
- Place in the chilled metal block (or on ice) for 2 minutes.
- Pipette 180 μl of room temperature SOC into each well.
- Place at 37°C for 60 minutes. Shaking is not necessary.
- Warm selection plates to 37°C.
- Mix the cells thoroughly by pipetting, then perform several 10-fold
serial dilutions in SOC.
- Spread 50-100 μl of each dilution onto a selection plate and incubate
overnight at 37°C.
Cells are best thawed in chilled metal 96-well block and DNA
added as soon as cells thawed (less than 2 minutes).
Incubation of DNA with Cells on Ice: For maximum transformation
efficiency, cells and DNA should be incubated together on ice for 20
minutes. Expect a 2-fold loss in transformation efficiency when this step is
shortened to 2 minutes.
Both the temperature and the timing of the heat shock step
are important and specific to the transformation volume and vessel. Using
the plate provided, 10 seconds at 42°C is optimal.
Outgrowth at 37°C for 1 hour is best for cell recovery and for
expression of antibiotic resistance. Expect a 2-fold loss in transformation
efficiency for every 15 minutes this step is shortened. SOC gives 2-fold
higher transformation efficiency than LB medium. Shaking is not necessary.
Selection plates can be used warm or cold, wet or dry without
significantly affecting the transformation efficiency. However, warm, dry
plates are easier to spread and allow for the most rapid colony formation.