User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/16: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==PCR mutation== | ==PCR mutation== | ||
* | * In reference to the [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|PCR Mutation protocol]], 100 ng/μL of the primer was needed for the reaction. The weight of the primer in the provided container was [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/10|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 | |||
<math>\frac{0.46E6 ng}{x \mu L}</math> = <math>\frac{100 ng}{1 \mu L}</math> 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 M<sub>1</sub>V<sub>1</sub> = M<sub>2</sub>V<sub>2</sub>, 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. | |||
V<sub>1</sub> = <math>\frac{100 ng/ \mu L * 1000 \mu L}{0.46E3 ng/ \mu L}</math> = 217.39 μL of the dissolved primer in water | |||
* The procedure listed in [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|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 [http://openwetware.org/wiki/Image:PHofluminol.pdf 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: | |||
<math>\frac{1.91 g}{15 mL}</math> × <math>\frac{1 mol}{105.9784 g}</math> = <math>\frac{0.00120 mol}{mL}</math> × <math>\frac{1 mL}{1E(-3) L}</math> = <math>\frac{1.20 mol}{L}</math> = 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 = <math>\frac{10 mM * 6 mL}{21 mL}</math> = 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. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''' | |||
| align="center" style="background:#f0f0f0;"|''HRP'' | |||
| align="center" style="background:#f0f0f0;"|''4-iodophenol'' | |||
| align="center" style="background:#f0f0f0;"|''luminol'' | |||
| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O<sub>2</sub>'' | |||
| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O'' | |||
|- | |||
| 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 | |||
|- | |||
Latest revision as of 22:07, 26 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||||||||
PCR mutation
0.46 mg = 0.46E6 ng
Continuation of Chemiluminescence
[math]\displaystyle{ \frac{1.91 g}{15 mL} }[/math] × [math]\displaystyle{ \frac{1 mol}{105.9784 g} }[/math] = [math]\displaystyle{ \frac{0.00120 mol}{mL} }[/math] × [math]\displaystyle{ \frac{1 mL}{1E(-3) L} }[/math] = [math]\displaystyle{ \frac{1.20 mol}{L} }[/math] = 1.20 M of sodium carbonate
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