Team:NYMU-Taipei/Notebook-Protocol

protocol

protocol


E.coli protocol


Two-day efficient cloning cycle


We used an efficient two day cloning cycle split into a "Light" day and a "Heavy" day.

Light Day


The light day consists of Colony PCR and liquid culture of colonies transformed from a previous day.


1. The 3-in-1


First, count the number of colonies you want to check. Then, do the following 3 things sequentially:

  • Liquid culture

  • 2nd time plate

  • Colony PCR

    • (Use the same tip to add the template to these three things)

    2. Make the gel for electrophoresis


    3. Run gel electrophoresis to check the colony PCR product


    Heavy Day


    The heavy day consists of:

  • Previously grown plasmid extraction

  • Plasmid PCR

  • Gel extraction

  • Digestion

  • Ligation

  • Transformation

    • Two-day efficient cloning cycle


    We are well aware of the skin, eye, and respiratory tract irritation E. coli may cause, therefore, all members of our wet-lab team are required to wear full personal protection gear when operating with E. coli competent cells and cultures. All materials that came in contact with bacterial cultures or competent cells were autoclaved and disposed properly.

       
       
       
       
       
       
       

    Two-day efficient cloning cycle


    We are well aware of the skin, eye, and respiratory tract irritation E. coli may cause, therefore, all members of our wet-lab team are required to wear full personal protection gear when operating with E. coli competent cells and cultures. All materials that came in contact with bacterial cultures or competent cells were autoclaved and disposed properly.