Team:Korea U Seoul/Project/Protocols

From 2013.igem.org

Revision as of 22:45, 27 September 2013 by Unjgs (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

CaCO3 Precipitation Assay

Method

The overall reaction for precipitation is

Ca2+ + HCO3 - → CaCO3 + H+


According 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.