McClean:Transformation of E. Coli

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Contents

Overview

Our lab's protocol for transforming E. coli competent cells prepared using the Inoue method.

Materials

  • E. Coli Competent Cells (See E. Coli Competent Cell Protocol)
  • Plasmid DNA to Transform
  • Selection PLates: LB/ampicillin or LB/Kan or other appropriate antibiotic containing plates (See Media Page)
  • LB Medium
  • Sterile H2O

Instructions

  1. Aliquot 10ng of plasmid DNA into a volume of 10 to 25 µl into a sterile 15-ml round-bottom test tube and place on ice.
  2. Rapidly thaw competent cells by warming between hands and dispense 50 µl immediately into test tubes containing DNA. Gently swirl tubes to mix, then place on ice for 10 min. (Competent cells should be used immediately after thawing. Remaining cells cannot be reused and should be discarded)
  3. Heat shock cells by placing tubes into 42°C water bath for 2 min. (Alternatively incubate at 37°C for 5 minutes)
  4. Add 1ml LB medium to each tube. Place each tube on a roller drum at 250 rpm for 1hr at 37°C (If you are doing only one plasmid this step is optional)
  5. Plate aliquots of transformation culture on LB/ampicillin or other appropriate antibiotic containing plates. (It is convenient and wise to plot several different dilutions of each transformation culture. The remainder of the mixture can be stored at 4°C for subsequent platings. One method is to plate 200 µl into one plate and a few drops into another plate).
  6. When plates are dry, incubate for 12-16 hrs at 37°C

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
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  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Current Protocols in Molecular Biology. V1 Section 1.8.2

Contact

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