High Efficiency Transformation Protocol with NEB 10-beta in 96-well Plate Format (NEB# C3019P)

  1. Chill a metal 96-well block on ice.

  2. 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.

  3. Carefully remove the aluminum foil seal from the plate or pierce holes through the foil seal with pipette tips.

  4. 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.

  5. Seal the plate with plate cover, or cap strips, or tapes.

  6. Incubate the plate in the chilled metal block (or on ice) for 20 minutes.

  7. 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.

  8. Place in the chilled metal block (or on ice) for 2 minutes.

  9. Pipette 180 μl of room temperature NEB 10-beta/Stable Outgrowth Medium into each well.

  10. Place at 37°C for 60 minutes. Shaking is not necessary.

  11. Warm selection plates to 37°C.

  12. Mix the cells thoroughly by pipetting, then perform several 10- fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.

  13. Spread 50–100 μl of each dilution onto a selection plate and incubate overnight at 37°C.