Biomolecular Breadboards:Protocols:Reaction Preparation

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Typical Reaction Preparation

This is just a typical reaction preparation. Modify it according to the designed experiment.

  • 90 μL is considered 1 reaction volume.
  • 90% of reaction is prepared (81 μL):
    • keep the extract tube on ice to start the reaction at the designed time: gene expression is not supposed to start at 0°C
    • add components in the extract tubes
    • add components in the order of ingredients from top to bottom
    • mix well whenever one component is added
  • Aliquot 90% of reaction by 10.8μl
  • Add 1.2μl of IPTG or ATc at 10x
    • i.e. add 1.2μl of 500μM of IPTG to fix 50μM of IPTG at final concentration
  • Incubate samples in incubator at 29°C overnight or run samples in plate reader at 29°C

Example Reaction:

Ingredient: 90%

  • Extract: 30μl
  • Water: 10.77μl
  • Mg-­‐glutamate at 100mM (1mM at final): 0.9μl
  • K-­‐glutamate at 3M (40mM at final): 1.2μl
  • Amino acids at 6mM (1.5mM at final): 22.5μl
  • 3-­‐PGA buffer 14x: 6.43μl
  • DTT at 100mM (1mM at final): 0.9μl
  • PEG8000 at 40% (2% at final): 4.5μl
  • pBEST-­‐p15A-­‐PL-­‐tetO1-­‐UTR1-­‐LacI-­‐T500 at 100nM (1nM at final): 0.9μl
  • pBEST-­‐p15A-­‐PL-­‐tetO1-­‐UTR1-­‐Venus-­‐T500 at 90nM (1nM at final): 1μl
  • pBEST-­‐p15A-­‐PL-­‐lacO1-­‐UTR1-­‐TetR-­‐T500 at 100nM (1nM at final): 0.9μl
  • pBEST-­‐p15A-­‐PL-­‐lacO1-­‐UTR1-­‐deCFP-­‐T500 at 90nM (1nM at final): 1μl

Preparation:

  • Aliquot it by 10.8μl and add 1.2μl of IPTG or anhydrotetracycline at 10x (50μM or 5μg/ml at final, respectively)
  • For end-‐point histogram, incubate them at 29°C overnight
  • For kinetics, run them in plate reader at 29°C
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