IGEM:Harvard/2010/General Protocols

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Contents

Growing a Bacterial E. coli Culture

E. coli is a typical bacteria used for molecular cloning. Long-term storage of BioBrick plasmids is in E. coli suspended in glycerol (500ul cells:500ul glycerol) and kept at -80˚C. Growing E. coli is performed at 37˚C, at which temperature they divide every 20 minutes.

  • Day1: Making streaks from glycerol stocks
    • Warm agar plate at 37˚C for 10 minutes
    • Label lid of plate with what you're streaking on it, and your name, and the date
    • Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
    • Incubate plate overnite at 37˚C
  • Day2: Growing liquid cultures

Plasmid Minipreps: Qiagen Miniprep Kit

To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.

DNA Digests

DNA Gel Electrophoresis and Fragment Purification

Measuring DNA Concentration using a Nanodrop Spectrophotometer

Ligation Reaction

Transformation of E. coli

Confirming the Assembly

Growing Arabidopsis

Growing an Agrobacterium Culture

Arabidopsis transformation via Flower Dipping

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