Team:Washington/Protocols/MMAssay
From 2010.igem.org
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'''Prepare a 10x Master Mix with these concentrations (10uL/rxn)''' | '''Prepare a 10x Master Mix with these concentrations (10uL/rxn)''' | ||
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Make one for each enzyme | Make one for each enzyme | ||
*HEPES 7.4 pH (250mM) | *HEPES 7.4 pH (250mM) |
Revision as of 18:14, 25 October 2010
Michaelis-Menten Assay
Prepare a 10x Master Mix with these concentrations (10uL/rxn)
Make one for each enzyme
- HEPES 7.4 pH (250mM)
- 1% Tween
- Purified Enzyme (50nM)
Note: Make one without enzyme for blank
- Dilute enzyme in 1x HEPES/Tween buffer
Dilute Substrate
- Prepare 10x substrate (10uM) in one well of a 12-well strip tube
- Do half concentration serial dilutions until 11th well.
- Leave the 12th well with no substrate
1.1x Master Mix
- Put equal parts 10x Master Mix and 10x L-Glu eppendorf tube (Referred to as L-Glu Master Mix)
- Put equal parts 10x Master Mix and diH2O in an eppendorf tube (Referred to as diH2O Master Mix)
- Fill each tube with diH2O till 90uL/rxn is reached
Prepare a 96-well plate for transpeptidation
- Pipette 90uL of 1.1x L-Glu Master Mix for one each enzyme across a row for the transpeptidation reaction
- Repeat for each enzyme
- Make one row of blank (1.1x L-Glu Master Mix without enzyme)
Prepare a 96-well plate for hydrolysis
- Pipette 90uL of 1.1 diH2O Master Mix for each different enzyme in each new row
- Repeat for each enzyme
- Make one row of blank (1.1x diH2O Master Mix without enzyme)
Final Concentrations
- Enzyme (CapD) - 5nM (0.00025mg/mL)
- Amino Acid (L-Glutamate) - 0mM or 5mM
- Substrate - Variable Concentration
- HEPES (7.4pH) - 25mM
- 0.1% Tween
Final Reaction Volume 100uL