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

PCR mutation

• 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

= 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 = = 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.

Continuation of Chemiluminescence

• 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:

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