From 2007.igem.org

• Home
  • Site Map
  • Meet the Team
  • Fun with iGEM
  • IcGEMs@OpenWetware
  • Acknowledgements
• Infector Detector
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • F2620 Comparison
  • Conclusion
• Cell by Date
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • Conclusion
• Cell-Free
  • What is Cell-Free?
  • Cell-Free Vs. Cell
  • Our Contributions
  • Characterisation
  • Applications
• Wet Lab
  • Lab Notebook
  • Protocols & Results
  • DNA Constructs
• Dry Lab
  • Modelling
  • Data Analysis
  • Software

Using the commercial E. Coli cell extract

Aims

Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega.

Equipment

• Eppendorf Tubes
• Gilson p20,p200,p1000

Reagents

• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Nuclease Free water x1ml
• DNA from midiprep/maxiprep

Protocols

1. From the commercially available kit, one reaction mixture comprises of:
   • 2.5ul of two different types of amino acid mixtures (total volume of 5ul)
   • 15ul of S30 Extract, Circular
   • 20ul of S30 Premix without amino acids

This makes up a total of 40ul.

1. Add the appropriate amount of DNA to the reaction mixture. (For our experiments we use 2ug and 4ug worth of DNA.)
2. Top up the mixture to 60ul with nuclease free water.