User:Allison K. Alix/Notebook/Thesis Research/2013/04/16

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Gel Electrophoresis/Abs/thiol-DNA-ThT Rxn</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Procedures==
==Procedures==
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Part one: Reconstituting samples in water
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Part one: Reconstituting DNA samples in water
1) Remove epi-test tubes from lyophilizer
1) Remove epi-test tubes from lyophilizer
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Part 2: Gel Electrophoresis.
Part 2: Gel Electrophoresis.
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1) Follow procedure outlined on  
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1) Follow procedure outlined on [[User:Allison K. Alix/Notebook/Thesis Research/2013/03/29|03/29/2013]]
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53.1micM DNA x = 500micL (5.82micM)
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2) Prepare the following 7 samples:
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54.8micL in 445.2 mic buffer
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* 1μL 1kb DNA ladder + 1μL loading dye + 4μL filtered water
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* 1μL 100bp DNA ladder + 1μL loading dye + 4μL filtered water
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 +
* 1μL thiol-DNA/AuNP + 1μL loading dye + 4μL filtered water
 +
 +
* 1μL supernatant 1 + 1μL loading dye + 4μL filtered water
 +
 +
* 1μL supernatant 2 + 1μL loading dye + 4μL filtered water
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 +
* 1μL supernatant 3 + 1μL loading dye + 4μL filtered water
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 +
* 1μL supernatant 4 + 1μL loading dye + 4μL filtered water
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 +
3) Load into gel
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 +
4) Stain with ehtidium bromide
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 +
5) Observe under UV Light
 +
 +
Part 3: Preparing thiol-DNA/ThT solution
 +
 +
* Because we are reacting the ThT with the DNA prior to reaction with the gold nanoparticles, it is important to account for an extra dilution that minimizes the concentration of the DNA. We are looking for the DNA to be 75x higher than the concentration of the gold nanoparticles (19.4nM x 74 = 1455nM). In order to have this concentration of DNA by the time it is ready to be mixed with the AuNP, it was calculated that we need to start with a 5.82μM solution
 +
 +
1) Dilute stock DNA (53.1μM) to 5.82μM
 +
 +
53.1μM DNA (x) = (500μL) (5.82μM)
 +
 +
X = 54.8micL in 445.2 mic buffer
 +
 +
2) Mix 250μL thiol-DNA with 250μL 5μMThT
 +
 +
3) Heat at ~75°C for 25 minutes
 +
 +
4) Cool to room temperature
 +
 +
==Data==
 +
 +
[[Image:Thiol-DNA_ThT_AuNP_absorbance.png]]
 +
 +
==Observations==
 +
 +
The absorbance data did not show a trend. The thiol-DNA/ThT/AuNP sample itself was the faintest shade of purple, which may be one reason we did not observe a distinguishable peak. Because of this, we decided to re-do the thiol-DNA/ThT reaction using a higher concentration of DNA (See Part 3 of the procedures). This allowed us to work with a higher concentration of gold nanoparticles since it's concentration is based off of the concentration of DNA used.
 +
 +
==Notes==
 +
 +
* We are still having trouble observing the gel under UV light. It is difficult to even see streaks due to the ladders. Because of this, we are going to focus on making a gel using different volumes of the ladders.
 +
 +
* combine complementary strand to see if that displaces the ThT

Current revision

Gel Electrophoresis/Abs/thiol-DNA-ThT Rxn Main project page
Previous entry      Next entry

Objectives

  • Reconstitute lyophilized DNA samples in 10 μL water and run gel.
  • Take absorbance spectra of thiol-DNA/ThT/AuNP sample and supernatants
  • Prepare thiol-DNA/ThT solution with higher concentrations of DNA

Procedures

Part one: Reconstituting DNA samples in water

1) Remove epi-test tubes from lyophilizer

2) Add 10 μL water to each sample and vortex to dissolve

Part 2: Gel Electrophoresis.

1) Follow procedure outlined on 03/29/2013

2) Prepare the following 7 samples:

  • 1μL 1kb DNA ladder + 1μL loading dye + 4μL filtered water
  • 1μL 100bp DNA ladder + 1μL loading dye + 4μL filtered water
  • 1μL thiol-DNA/AuNP + 1μL loading dye + 4μL filtered water
  • 1μL supernatant 1 + 1μL loading dye + 4μL filtered water
  • 1μL supernatant 2 + 1μL loading dye + 4μL filtered water
  • 1μL supernatant 3 + 1μL loading dye + 4μL filtered water
  • 1μL supernatant 4 + 1μL loading dye + 4μL filtered water

3) Load into gel

4) Stain with ehtidium bromide

5) Observe under UV Light

Part 3: Preparing thiol-DNA/ThT solution

  • Because we are reacting the ThT with the DNA prior to reaction with the gold nanoparticles, it is important to account for an extra dilution that minimizes the concentration of the DNA. We are looking for the DNA to be 75x higher than the concentration of the gold nanoparticles (19.4nM x 74 = 1455nM). In order to have this concentration of DNA by the time it is ready to be mixed with the AuNP, it was calculated that we need to start with a 5.82μM solution

1) Dilute stock DNA (53.1μM) to 5.82μM

53.1μM DNA (x) = (500μL) (5.82μM)

X = 54.8micL in 445.2 mic buffer

2) Mix 250μL thiol-DNA with 250μL 5μMThT

3) Heat at ~75°C for 25 minutes

4) Cool to room temperature

Data

Image:Thiol-DNA_ThT_AuNP_absorbance.png

Observations

The absorbance data did not show a trend. The thiol-DNA/ThT/AuNP sample itself was the faintest shade of purple, which may be one reason we did not observe a distinguishable peak. Because of this, we decided to re-do the thiol-DNA/ThT reaction using a higher concentration of DNA (See Part 3 of the procedures). This allowed us to work with a higher concentration of gold nanoparticles since it's concentration is based off of the concentration of DNA used.

Notes

  • We are still having trouble observing the gel under UV light. It is difficult to even see streaks due to the ladders. Because of this, we are going to focus on making a gel using different volumes of the ladders.
  • combine complementary strand to see if that displaces the ThT




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