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

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(PCR mutation)
Current revision (10:00, 7 December 2012) (view source)
(Continuation of Chemiluminescence)
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{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''
| align="center" style="background:#f0f0f0;"|''HRP''
| align="center" style="background:#f0f0f0;"|''HRP''
| align="center" style="background:#f0f0f0;"|''4-iodophenol''
| align="center" style="background:#f0f0f0;"|''4-iodophenol''
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| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O''
| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O''
| 33 μL||27 μL||450 μL||88 μL||902 μL
| 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

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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

\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 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:

\frac{1.91 g}{15 mL} × \frac{1 mol}{105.9784 g} = \frac{0.00120 mol}{mL} × \frac{1 mL}{1E(-3) L} = \frac{1.20 mol}{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 = \frac{10 mM  *  6 mL}{21 mL} = 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 solutions2.3 μM18 mM10 mM1.7 mMneutral
Volume33 μL27 μL450 μL88 μL902 μL

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