# Difference between revisions of "User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/16"

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## 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

${\displaystyle {\frac {0.46E6ng}{x\mu L}}}$ = ${\displaystyle {\frac {100ng}{1\mu L}}}$ 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 = ${\displaystyle {\frac {100ng/\mu L*1000\mu L}{0.46E3ng/\mu L}}}$ = 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 amplified DNA was contained in a sterilized, 1.5 microcentrifuge tube.

## 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 final pH for luminol was 10.55.
• The molarity of sodium carbonate (MW 105.9784 g/mol) added was calculated:

${\displaystyle {\frac {1.91g}{15mL}}}$ × ${\displaystyle {\frac {1mol}{105.9784g}}}$ = ${\displaystyle {\frac {0.00120mol}{mL}}}$ × ${\displaystyle {\frac {1mL}{1E(-3)L}}}$ = ${\displaystyle {\frac {1.20mol}{L}}}$ = 1.20 M of sodium carbonate

• The concentration of luminol has been diluted by the addition of 15 mL of water.

Molarity of diluted luminol = ${\displaystyle {\frac {10mM*6mL}{21mL}}}$ = 2.85714 mM of luminol

• Since there was limited amounts of the solid form of luminol, it was decided to take 6 mL of the 2.86 mM luminol stock solution and then add the appropriate amount of luminol to the 6 mL volume. The molarity of the 6 mL solution was very minute; the molarity was approximated as 2 mM. By making this assumption, .00106 g of luminol would make a 1 mM solution in 6 mL of water; this amount was multiplied by 8. The product was 0.00848 g of luminol was needed to be added into the 2 mM solution of luminol to increase the molarity to 10 mM. The amount was weighed and added to the 6 mL 2.86 mM solution of luminol.
• Due to time constraints, the chemiluminescence of the luminol at pH 10.55 was tested on a lab bench with the room lights turned off. The reaction produced a neon blue glow that lasted for more than 3 minutes. No photograph was taken since the apparatus of the camera was not suited for the fluorescence activity.
• The volume of each reagent added to the cuvette are listed below.
 ' HRP 4-iodophenol luminol H2O2 H2O Molarity of stock solutions 2.3 μM 18 mM 10 mM 1.7 mM neutral Volume 33 μL 27 μL 450 μL 88 μL 902 μL