Home Protocols 5 Minute Transformation Protocol using NEB10-beta Competent E. coli  (C3019H/C3019I)

5 Minute Transformation Protocol using NEB®10-beta Competent E. coli  (C3019H/C3019I)

Overview

The following protocol results in only 10% efficiency compared to the High Efficiency Transformation Protocol.

Protocol

  1. C3019H: Remove cells from -80°C freezer and thaw in your hand.
    C3019I: Thaw a tube of NEB 10-beta Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place the mixture on ice for 2 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 2 minutes. Do not mix.
  6. Pipette 950 µl of room temperature NEB® 10-beta/Stable Outgrowth Medium into the mixture. Immediately spread 50-100 µl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.