User:Allison K. Alix/Notebook/Thesis Research/2013/08/12

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(AuNP@SiO2/DNA attachment)
 
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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;"> Au@SiO2 DNA attachment</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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* AuNP@SiO2/DNA attachment procedure  (10X diluted AuNP@SiO2 prepared with 2uL TES to be used)
* AuNP@SiO2/DNA attachment procedure  (10X diluted AuNP@SiO2 prepared with 2uL TES to be used)
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==AuNP@SiO2/DNA attachment==
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adapted from Madeleine Y. Bee[http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/10]
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1) Prepare a fresh 10X dilution of AuNP@SiO2
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* Sonicate AuNP@SiO2 for 10 minutes
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* Mix 15uL AuNP@SiO2 with 135uL filter milliQ water (@ room temperature), vortex to mix thorooughly
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2) Mix 150uL AuNP@SiO2 with 15uL APS and 235uL 100% ethanol. Sonicate for 30 minutes
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3) Centrifuge and rinse solution a total of 5 times at 9100rpm for 5 minutes. Redisperse in ethanol for the first three and 10mM sodium phosphate buffer (pH=9) for the last two.
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NOTE: after first round of centrifugation, it was apparent 5 minutes was a sufficient amount of time. However, after removal of the supernatant and redispersion in ethanol, there was difficulty getting the particles back into solution. They were allowed to sonicate for 10 minutes to try to induce separation but still looked relatively aggregated. To further induce separate, the sample was subjected to ultrasonic liquid processing for ~15 seconds, which proved to be effective. The sample was only rinsed with phosphate buffer once, as it would not separate even after long periods of centrifugation '''Change centrifugation time to 30 seconds.'''
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4) Dilute 2mg Sulfo-SSMC with 200uL sodium phosphate buffer. Transfer to a larger epi-test tube and continue to dilute until total volume reaches 800uL. Vortex to completely dissolve.
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5) Mix 400uL Sulfo-SSMC with 400uL AuNP@SiO2/NH2 solution. Sonicate 1 hour.
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6) While solution is sonicating. Reduce S-DNA to HS-DNA. Mix 50uL 40uM S-DNA with 50uL DTT in TEA solution. React 10 minutes and extract DTT using ethyl acetate.
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NOTE: this is not enough volume to retain 50uL DNA after extraction. Use 250uL of DNA and 250uL DTT instead.
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Procedure was stopped at this point for the following reasons:
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1) The AuNP@SiO2 in sulfo SSMC was barely colored, leading me to believe that there were very few nano-particles in solution. I diluted the AuNP@SiO2 originally because there was not 150uL of the concentrated solution available. I believe this procedure will work best using 150uL of the concentrated sample. It is centrifuged many times and after every centrifugation period, there is a chance particles are being lost.
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2) I was not able to rinse twice with SPB. I could not get the particles to separate (as previously noted) after my first rinse with SPB. This was concerning and led me to believe there were very few AuNP in solution. I want to be sure there are the maximum number of particles available for the DNA attachment.
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* I have decided to prepare a new batch of silica coated AuNP. The MHA/AuNP solution will stir between tonight and tomorrow morning, at which point I can add the TES and begin the DNA attachment procedure again (This should all be completed by tomorrow)
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Current revision

Au@SiO2 DNA attachment Main project page
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To Do

  • AuNP@SiO2/DNA attachment procedure (10X diluted AuNP@SiO2 prepared with 2uL TES to be used)

AuNP@SiO2/DNA attachment

adapted from Madeleine Y. Bee[1]

1) Prepare a fresh 10X dilution of AuNP@SiO2

  • Sonicate AuNP@SiO2 for 10 minutes
  • Mix 15uL AuNP@SiO2 with 135uL filter milliQ water (@ room temperature), vortex to mix thorooughly

2) Mix 150uL AuNP@SiO2 with 15uL APS and 235uL 100% ethanol. Sonicate for 30 minutes

3) Centrifuge and rinse solution a total of 5 times at 9100rpm for 5 minutes. Redisperse in ethanol for the first three and 10mM sodium phosphate buffer (pH=9) for the last two.

NOTE: after first round of centrifugation, it was apparent 5 minutes was a sufficient amount of time. However, after removal of the supernatant and redispersion in ethanol, there was difficulty getting the particles back into solution. They were allowed to sonicate for 10 minutes to try to induce separation but still looked relatively aggregated. To further induce separate, the sample was subjected to ultrasonic liquid processing for ~15 seconds, which proved to be effective. The sample was only rinsed with phosphate buffer once, as it would not separate even after long periods of centrifugation Change centrifugation time to 30 seconds.

4) Dilute 2mg Sulfo-SSMC with 200uL sodium phosphate buffer. Transfer to a larger epi-test tube and continue to dilute until total volume reaches 800uL. Vortex to completely dissolve.

5) Mix 400uL Sulfo-SSMC with 400uL AuNP@SiO2/NH2 solution. Sonicate 1 hour.

6) While solution is sonicating. Reduce S-DNA to HS-DNA. Mix 50uL 40uM S-DNA with 50uL DTT in TEA solution. React 10 minutes and extract DTT using ethyl acetate.

NOTE: this is not enough volume to retain 50uL DNA after extraction. Use 250uL of DNA and 250uL DTT instead.

Procedure was stopped at this point for the following reasons:

1) The AuNP@SiO2 in sulfo SSMC was barely colored, leading me to believe that there were very few nano-particles in solution. I diluted the AuNP@SiO2 originally because there was not 150uL of the concentrated solution available. I believe this procedure will work best using 150uL of the concentrated sample. It is centrifuged many times and after every centrifugation period, there is a chance particles are being lost.

2) I was not able to rinse twice with SPB. I could not get the particles to separate (as previously noted) after my first rinse with SPB. This was concerning and led me to believe there were very few AuNP in solution. I want to be sure there are the maximum number of particles available for the DNA attachment.

  • I have decided to prepare a new batch of silica coated AuNP. The MHA/AuNP solution will stir between tonight and tomorrow morning, at which point I can add the TES and begin the DNA attachment procedure again (This should all be completed by tomorrow)





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