"
Team:Washington/Protocols/MMAssay
From 2010.igem.org
(Difference between revisions)
(New page: {{Template:Team:Washington/Templates/Header}} <html> <script type="text/javascript"> $(function() { var mainimg = document.getElementById("mainimg"); var default_src = mainimg.src; v...) |
|||
| Line 25: | Line 25: | ||
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | <!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | ||
='''Michaelis-Menten Assay'''= | ='''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 | ||
| + | |||
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------> | <!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------> | ||
Revision as of 18:12, 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
