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Team:Korea U Seoul/Project/sub proc result
From 2013.igem.org
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<h2>Procedure</h2> | <h2>Procedure</h2> | ||
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| - | <li>Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. | + | <li>Blank Determination</li> |
| - | + | Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. | |
| + | Withdraw in a 5 ml syringe, 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0. | ||
| + | <li>Enzyme Determination</li> | ||
| + | Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T. | ||
</ul> | </ul> | ||
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Revision as of 21:15, 27 September 2013
CaCO3 Precipitation Assay
Method
The overall reaction for precipitation isAccording to that equation, CaCO3 generated the more the pH will be lowered.
The effect of NA and R on the rate of precipitation of CaCO3 was determined by recording the decrease in pH of a 20mM Tris HCl pH8.0 buffer solution at 10°C
Reagents
- 20mM Tris-HCl buffer, pH 8.0.
- 20mM Tris-HCl buffer, pH 8.0 + 1M Imidazole.
- 1M NaHCO3.
- 1M CaCl2.
Enzyme
Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.
Procedure
- Blank Determination Add 0.5 ml of 1M CaCl2 to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Immediately start a stop watch and record the time and pH up to 20 minutes. - Enzyme Determination Add freshly diluted enzyme to a 15 ml tube cotaining 0.5 ml of 1M NaHCO3 and 9 ml of 20mM Tris-HCl buffer. Add Add 0.5 ml of 1M CaCl2. Immediately start a stop watch and record the time and pH up to 20 minutes.
Carbonic Anhydrase Assay
Method
The electrometric method of Wilbur and Anderson (1948) in which the time required (in seconds) for a saturated CO2 solution to lower the pH of 0.012 M Tris⋅HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T0; with enzyme, T.A unit of activity = (2×(T_0-T))/T
Reagents
0.02 M Tris⋅HCl buffer, pH 8.0. Store in an ice bath at 0-4°C before and during use.Carbon dioxide saturated water. Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath.
Enzyme
Using Protino® Ni-NTA Columns for His-tag protein purification, purify the enzymes.These enzymes is diluted in 20mM Tris-HCl buffer, pH 8.0 + 10mM Immidazole at 4°C.
Procedure
- Blank Determination Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Withdraw in a 5 ml syringe, 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0.
- Enzyme Determination Add 6.0 ml of 20 mM Tris⋅HCl buffer, pH 8.0 to a 15ml tube. Maintain temperature at 0-4°C and record pH. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.
