User:Allison K. Alix/Notebook/Thesis Research/2013/05/14: Difference between revisions
(→Data) |
|||
| Line 108: | Line 108: | ||
In the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point. | In the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point. | ||
* Note that for the gel electrophoresis, if using the blue/orange loading dye, allow the gel to run until the lightest blue dye is 3/4 of the way across, this should be the dye that travels the slowest. (Overall took about ~2 hours) | |||
Revision as of 11:33, 14 May 2013
 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> | ||||||||||||||||||||||||
Objectives
Procedures
Previous concentrations used:
To be used:
Calculations: Preparing 17.8μM thiol-DNA original concentration of thiol-DNA- 53.1μM 53.1μM (x) = (17.8μM)(250μL) x= 83.8μL in 166.2μL buffer Preparing 20μM ThT original conc ThT= 5mM 5mM (x) = (0.02mM)(10mL) x=40μL in 9960μL H2O
1) Mix 250μL 17.8μM thiol-DNA with 250μL 20μM ThT
2) Heat at 75°C for ~25min 3) Cool to room temperature 4) Take UV-Vis/Fluorescence measurements of thiol-DNA/ThT sample Part 2: Hybridizing thiol-DNA/ThT with AuNP 1) Mix 250 μL 8.9μM thiol-DNA/10μM ThT solution with 250μL 4%TEA containing 100mM DTT
2) Extract DTT using 4 2mL aliquots of ethyl acetate 3) Mix 232.4μL of thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL sodium citrate buffer
Part 3: Gel Electrophoresis (2% Agarose) Prepare the following: 1) 5μL 100bp ladder with 1μL loading buffer 2) 5μL thiol-DNA/ThT/AuNP with 1μL loading buffer 3) 5μL supernatant 1 with 1μL loading buffer 4) 5μL supernatant 2 with 1μL loading buffer 5) 5μL supernatant 3 with 1μL loading buffer Data
ObservationsIn the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point.
| |||||||||||||||||||||||||
