User:Allison K. Alix/Notebook/Thesis Research/2013/05/29: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> DNA/AuNP/MB Preparation</span> | ||
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* Prepare DTT/TEA solution to be used in DNA extraction procedures. | * Prepare DTT/TEA solution to be used in DNA extraction procedures. | ||
* Prepare DNA/AuNP mixture | |||
* Bind MB to DNA/AuNP | |||
==Procedures== | |||
Part 1: Preparation of 100mM DTT in 4% TEA solution | |||
Original DTT concentration 1M | |||
1M(x) = (100x10<sup>-3</sup>M)(1mL) | |||
x = 0.1mL = 100μL in 900μL 4% TEA | |||
4%(900μL) = 36μL TEA in 864μL PBS | |||
OVERALL: 100μL 1M DTT + 36μL TEA + 864μL PBS | |||
Part 2: Prepare DNA-AuNP to bind MB to | |||
1) Mix 100μL 2.91μM thiol-DNA with 100μL 100mM DTT in 4% TEA. React 10 minutes | |||
[DNA]=1.45μM | |||
2) Extract DTT using 4 2mL aliquots of ethyl acetate | |||
3) Mix 93μL thiol-DNA with 93μL 19.4nM AuNP and 14μL sodium citrate buffer. React 10 minutes. | |||
[DNA] = 727.5nM [AuNP] = 9.7nM | |||
4) Centrifuge and redisperse in 50mM HEPES buffer | |||
5) Mix 20μL of 7.61μM MB with 100μL of thiol DNA/AuNP mixture | |||
6) Heat @75°C for 25 minutes. | |||
7) Observe absorbance and fluorescence spectra | |||
Revision as of 12:36, 3 June 2013
 DNA/AuNP/MB Preparation
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Objectives
ProceduresPart 1: Preparation of 100mM DTT in 4% TEA solution Original DTT concentration 1M 1M(x) = (100x10-3M)(1mL) x = 0.1mL = 100μL in 900μL 4% TEA 4%(900μL) = 36μL TEA in 864μL PBS OVERALL: 100μL 1M DTT + 36μL TEA + 864μL PBS Part 2: Prepare DNA-AuNP to bind MB to 1) Mix 100μL 2.91μM thiol-DNA with 100μL 100mM DTT in 4% TEA. React 10 minutes [DNA]=1.45μM 2) Extract DTT using 4 2mL aliquots of ethyl acetate 3) Mix 93μL thiol-DNA with 93μL 19.4nM AuNP and 14μL sodium citrate buffer. React 10 minutes. [DNA] = 727.5nM [AuNP] = 9.7nM 4) Centrifuge and redisperse in 50mM HEPES buffer 5) Mix 20μL of 7.61μM MB with 100μL of thiol DNA/AuNP mixture 6) Heat @75°C for 25 minutes. 7) Observe absorbance and fluorescence spectra
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