User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/16: Difference between revisions
From OpenWetWare
No edit summary |
|||
(5 intermediate revisions by the same user not shown) | |||
Line 25: | Line 25: | ||
.56mg= 5600 ng | .56mg= 5600 ng | ||
M<sub>1</sub>V<sub>1</sub>= M<sub>2</sub>V<sub>2</sub> | M<sub>1</sub>V<sub>1</sub>= M<sub>2</sub>V<sub>2</sub> | ||
5600 ng/μL x V<sub>1</sub>= 100ng x 1000μL | 5600 ng/μL x V<sub>1</sub>= 100ng x 1000μL | ||
*(The container containing the plasmid only had room for 1mL of water to be added- as a result, a dilution needed to be done in order to get the appropriate concentration. | *(The container containing the plasmid only had room for 1mL of water to be added- as a result, a dilution needed to be done in order to get the appropriate concentration. 1mL of water was added to dissolve the plasmid prior to the dilution | ||
V<sub>2</sub>= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL | V<sub>2</sub>= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL | ||
Line 62: | Line 63: | ||
* a new stock of luminol solution was made at a pH of 10.56. For full details on preparation, calculation, and concentration please see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/16| Mary's Notebook]] | * a new stock of luminol solution was made at a pH of 10.56. For full details on preparation, calculation, and concentration please see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/16| Mary's Notebook]] | ||
* The other reagents were either left over from the HRP absorbance assay from [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02| another week]] or were remade in using the same method. The final concentrations and volumes are as follows. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''Desired concentration in cuvette''' | |||
| align="center" style="background:#f0f0f0;"|'''M1''' | |||
| align="center" style="background:#f0f0f0;"|'''V1''' | |||
| align="center" style="background:#f0f0f0;"|'''M2''' | |||
| align="center" style="background:#f0f0f0;"|'''V2''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''HRP''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Iodophenol||.18mM||10mM||27μL||.18mM||1500μL||||Concentration of compound in Cuvette||Volume | |||
|- | |||
| H202||.1mM||1.7mM||88μL||.1mM||1500μL||||20mM||30μL | |||
|- | |||
| Luminol||3mM||30mM||450μL||3mM||1500μL||||22mM||33μL | |||
|- | |||
| ||||||||||||||24mM||36μL | |||
|- | |||
| ||||||||||||||26mM||39μL | |||
|- | |||
| ||||||||||||||28mM||42μL | |||
|} | |||
==Calculations== | |||
*Concentrations of the stock solutions: | |||
**10mM iodophenol in Dimethyl Sulfoxide (DMSO) | |||
**1.7mM H<sub>2</sub>0<sub>2</sub> | |||
**30mM luminol | |||
**1mg/mL HRP | |||
Total Volume in cuvet= 1500μL | |||
==Conclusions== | ==Conclusions== | ||
* The goal for today was the run the HRP chemiluminescence assay. Last week there had been no emittance from the sample. The luminol pH had been at 8 and after researching the literature, we decided that it was necessary to increase the pH of luminol in order to push forward a reaction. As a result, we aimed to make a luminol solution with a pH of 10.5. The actual pH of the luminol was 10.56. | * The goal for today was the run the HRP chemiluminescence assay. Last week there had been no emittance from the sample. The luminol pH had been at 8 and after researching the literature, we decided that it was necessary to increase the pH of luminol in order to push forward a reaction. As a result, we aimed to make a luminol solution with a pH of 10.5. The actual pH of the luminol was 10.56. | ||
* We also decided that we needed to increase the concentration of the HRP in the cuvet. The Fluorimeter was not working and so we just used visual analysis to see if any fluorescence was occurring. The first concentration in the cuvet we tested was 2mM- which did not produce any luminescence. The next concentration we attempted was 10mM which also did not result in any luminescence. We then decided to double this final concentration to 20mM HRP while maintaining 3mM concentration of Luminol. This sample resulted in fluorescence of the luminol in the form of a blue light! We plan on running different variations of the HRP concentration beginning with 20mM tomorrow when conducting the chemiluminescence assay. | * We also decided that we needed to increase the concentration of the HRP in the cuvet. The Fluorimeter was not working and so we just used visual analysis to see if any fluorescence was occurring. The first concentration in the cuvet we tested was 2mM- which did not produce any luminescence. The next concentration we attempted was 10mM which also did not result in any luminescence. We then decided to double this final concentration to 20mM HRP while maintaining 3mM concentration of Luminol. This sample resulted in fluorescence of the luminol in the form of a blue light! We plan on running different variations of the HRP concentration beginning with 20mM tomorrow when conducting the chemiluminescence assay. All other reagents will remain constant. | ||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 16:43, 21 November 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
PCR of ADA MutationsGoals
Preparation
Concentration of plasmid needed for experiment= 100ng/μL
.56mg= 5600 ng M1V1= M2V2 5600 ng/μL x V1= 100ng x 1000μL
V2= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL Procedure
Calculations
Total Volume in cuvet= 1500μL Conclusions
|