High Efficiency Transformation Protocol with NEB 10-beta in 96-well Plate Format (NEB# C3019P)
- 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.
- 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 DNA.
- 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 NEB 10-beta/Stable Outgrowth Medium 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 NEB 10-beta/Stable Outgrowth Medium.
- Spread 50–100 μl of each dilution onto a selection plate and incubate overnight at 37°C.
