User:Floriane Briere/Notebook/CHEM-496/2012/02/15: Difference between revisions
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==Objective== | ==Objective== | ||
Today's objective is to perform a dialysis using the solution we made yesterday. The reaction has been running for 24hours in the shaker, allowing the dye to tag our BSA peptides by binding unprotonated lysine. The dialysis is going to allow us to remove the excess of dye which hasn't been able to react with our Gold NPs. This step | Today's objective is to perform a dialysis using the solution we made yesterday. The reaction has been running for 24hours in the shaker at room temperature, allowing the dye to tag our BSA peptides by binding unprotonated lysine. The dialysis is going to allow us to remove the excess of dye which hasn't been able to react with our Gold NPs (because number of available Lys site is limited). This step allows us to purify our solution so that we'll then be able to quantify labelled NPs using UV-Vis spectrum; and the efficiency of the dying process will be determined using fluorescence spectroscopy. | ||
== | The first step of today's experiment is to lower the pH of our solution so that the reaction stops; to do so, we have to neutralize Tris molecules using HCl (we have to add an equal amount of HCl to the amount of Tris we added yesterday). Then we'll perform a 7 days dialysis to purify our solution. We are going to save pH values at every step of the protocol because we believe this parameter is crucial. | ||
==Protocol== | |||
* Control solution (tube 1) | |||
# Add 16.8µL of HCl (0.948M) | |||
# Several minutes in the shaker | |||
# Check the pH using the pHmeter (SympHony): pH = 3.20 | |||
# Add 300µL of Tris buffer | |||
# Check the pH using the pHmeter (SympHony): pH = 6.49 | |||
* 166 ratio solution (tube 2) | |||
# Add 79µL of HCl (0.948M) | |||
# Several minutes in the shaker | |||
# Check the pH using the pHmeter (SympHony): pH = 2.72 | |||
# Add 1mL of Tris buffer | |||
# Check the pH using the pHmeter (SympHony): pH = 6.5 | |||
* 70 ratio solution (tube 3) | |||
# Add 32µL of HCl (0.948M) | |||
# Several minutes in the shaker | |||
# Check the pH using the pHmeter (SympHony): pH = 2.92 | |||
# Add 600µL of Tris buffer | |||
# Check the pH using the pHmeter (SympHony): pH = 6.09 | |||
* Run the dialysis into water, the water is going to be changed twice the next day (2/16/12) and then once a day until 2/21/12: | |||
# 2/16/12 (thursday) at 9:30am and 5:10pm | |||
# 2/17/12 (friday) at 2:15pm | |||
# 2/18/12 (saturday) at 1pm | |||
# 2/19/12 (sunday) at 2:45pm | |||
# 2/20/12 (monday) at 1:15pm | |||
The dialysis was performed using SnakeSkin® Dialysis Tubing (Thermo Scientific) | |||
==Data== | ==Data== | ||
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Concentration of Tris buffer = 50mM | Concentration of Tris buffer = 50mM | ||
Concentration of HCl solution = 0.948M | Concentration of HCl solution = 0.948M | ||
* Calculations for the control solution (tube 1) | |||
Volume of Tris buffer = 321µL | Volume of Tris buffer = 321µL | ||
=> Number of mole of Tris = 321 x 10^-6 x 50 x 10^-3 = 16µM | => Number of mole of Tris = 321 x 10^-6 x 50 x 10^-3 = 16µM | ||
=> Volume of HCl solution to add = (1.6 x 10^-5)/0.948 = 16.8µL | => Volume of HCl solution to add = (1.6 x 10^-5)/0.948 = 16.8µL | ||
* Calculations for the 166 ratio solution (tube 2) | |||
Volume of Tris buffer = 1.5mL | Volume of Tris buffer = 1.5mL | ||
=> Number of mole of Tris = 1.5 x 10^-3 x 50 x 10^-3 = 75µM | => Number of mole of Tris = 1.5 x 10^-3 x 50 x 10^-3 = 75µM | ||
=> Volume of HCl solution to add = (7.5 x 10^-5)/0.948 = 79µL | => Volume of HCl solution to add = (7.5 x 10^-5)/0.948 = 79µL | ||
* Calculations for the 70 ratio solution (tube 3). | |||
Volume of Tris buffer = 600µL | Volume of Tris buffer = 600µL | ||
=> Number of mole of Tris = 600 x 10^-6 x 50 x 10^-3 = 32µM | => Number of mole of Tris = 600 x 10^-6 x 50 x 10^-3 = 32µM | ||
=> Volume of HCl solution to add = (3.2 x 10^-5)/0.948 = 32µL | => Volume of HCl solution to add = (3.2 x 10^-5)/0.948 = 32µL | ||
==Notes== | ==Notes== | ||
* For the control solution (with HCl instead of HAuCl4; and with the same amounts as in the 166 ratio solution), the solution's color was a light gradient (from blue to purple); when we added the HCl the solution turned light pink. | |||
[[Image:15feb-Control solution.png]] | |||
* For the 166 ratio solution (initially the fibers' solution), when we removed it from the shaker it was blue with some dark fibers. When we added the HCl, the solution turned partially pink. | |||
[[Image:15feb-Fibers solution.png]] | |||
* For the 70 ratio solution (initially the purple solution), when we removed the solution from the shaker the solution's color was a dark gradient (from purple to blue); when we added the HCl the solution became lighter (with light blue and pink). | |||
[[ | [[Image:15feb-Purple solution.png]] | ||
Revision as of 11:46, 30 April 2012
 Chem-496
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ObjectiveToday's objective is to perform a dialysis using the solution we made yesterday. The reaction has been running for 24hours in the shaker at room temperature, allowing the dye to tag our BSA peptides by binding unprotonated lysine. The dialysis is going to allow us to remove the excess of dye which hasn't been able to react with our Gold NPs (because number of available Lys site is limited). This step allows us to purify our solution so that we'll then be able to quantify labelled NPs using UV-Vis spectrum; and the efficiency of the dying process will be determined using fluorescence spectroscopy. The first step of today's experiment is to lower the pH of our solution so that the reaction stops; to do so, we have to neutralize Tris molecules using HCl (we have to add an equal amount of HCl to the amount of Tris we added yesterday). Then we'll perform a 7 days dialysis to purify our solution. We are going to save pH values at every step of the protocol because we believe this parameter is crucial. Protocol
The dialysis was performed using SnakeSkin® Dialysis Tubing (Thermo Scientific) DataCalculations for the volume of HCl needed to neutralize Tris. Concentration of Tris buffer = 50mM Concentration of HCl solution = 0.948M
Volume of Tris buffer = 321µL => Number of mole of Tris = 321 x 10^-6 x 50 x 10^-3 = 16µM => Volume of HCl solution to add = (1.6 x 10^-5)/0.948 = 16.8µL
Volume of Tris buffer = 1.5mL => Number of mole of Tris = 1.5 x 10^-3 x 50 x 10^-3 = 75µM => Volume of HCl solution to add = (7.5 x 10^-5)/0.948 = 79µL
Volume of Tris buffer = 600µL => Number of mole of Tris = 600 x 10^-6 x 50 x 10^-3 = 32µM => Volume of HCl solution to add = (3.2 x 10^-5)/0.948 = 32µL Notes
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