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
