User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/16

(Difference between revisions)

Revision as of 02:30, 26 October 2012

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In reference to the PCR Mutation protocol, 100 ng/μL of the primer was needed for the reaction. The weight of the primer in the provided container was 0.46 mg. A ratio of the weight over volume was equated to the required concentration of the primer:

0.46 mg = 0.46E6 ng

$\frac{0.46E6 ng}{x \mu L}$ = $\frac{100 ng}{1 \mu L}$ of primer in water = 4600 μL of water

There is limited space in the plastic container (1 mL). Instead of dissolving 0.46E6 ng of the primer in 4600 μL of water, the entire primer was dissolved in 1 mL water.
Using M₁V₁ = M₂V₂, the volume taken from the solution of 0.46E6 ng in 1 mL of water was calculated to be 217.39 μL. This was transferred to a new tube and filled up with water to a total volume of 1 mL.

V₁ = $\frac{100 ng/ \mu L * 1000 \mu L}{0.46E3 ng/ \mu L}$ = 217.39 μL of the dissolved primer in water

The procedure listed in PCR Mutation protocol was strictly followed. After the addition of all reagents, the sample was placed in the thermocycler.

The luminol prepared from the previous chemiluminescence laboratory period had a pH of 7 to 8. Reviewing a journal article of Xiaoyu, it was determined that the optimal pH of luminol was 12.5. As a result, it was decided to prepare a new solution of luminol at a pH of 10 to 11.
A weight of .0112 g of luminol was added to 6 mL of water. The buffer composed of a direct addition of .0737 g of sodium carbonate and .4358 g of sodium bicarbonate.
Using a pH meter, the electrode detected the pH at 8.73. As suggested by Dr. Hartings, a solution of sodium carbonate was made to increase the pH of the solution.
Several adjustments were made in increasing the pH. A total weight of 1.91 g of sodium carbonate dissolved in 15 mL of water was added to the 6 mL solution of luminol.
The molarity of sodium carbonate (MW 105.9784 g/mol) added was calculated:

$\frac{1.91 g}{15 mL}$ × $\frac{1 mol}{105.9784 g}$

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 * Using a pH meter, the electrode detected the pH at 8.73. As suggested by Dr. Hartings, a solution of sodium carbonate was made to increase the pH of the solution.
 * Several adjustments were made in increasing the pH. A total weight of 1.91 g of sodium carbonate dissolved in 15 mL of water was added to the 6 mL solution of luminol.
 * The molarity of sodium carbonate (MW 105.9784 g/mol) added was calculated:
+<math>\frac{1.91 g}{15 mL}</math> × <math>\frac{1 mol}{105.9784 g}</math>