User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/10: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Silica-coating Reaction</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Silica-coating Reaction</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"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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====Procedure====
====Procedure====
*Combine 150μL AuNPs@SiO<sub>2</sub>, 15μL APS and 235μL ethanol
*Combine 150μL AuNPs@SiO<sub>2</sub>, 15μL APS, and 235μL ethanol
*Vortex solution for 30 minutes
*Sonicate solution for 30 minutes
*Rinse sample three times by centrifugation at 7700g (9081.99rpm) with ethanol and two times with sodium phosphate buffer, pH 9 (SPB)
*Rinse sample three times by centrifugation at 7700g (9081.99rpm) with ethanol and two times with sodium phosphate buffer, pH 9 (SPB)
*Make a separate solution of 1 mg sulfo-SMCC in 400μL of SPB
*Make a separate solution of 1 mg sulfo-SMCC in 400μL of SPB
*Add SSMCC solution to AuNP solution
*Add SSMCC solution to AuNP solution
*Vortex for 1 hour
*Sonicate for 1 hour
*Rinse twice with SPB
*Rinse by centrifugation at 7700g (9081.99rpm) twice with SPB
*Rinse twice with potassium phosphate buffer, pH 7.4 (KPB)
*Rinse by centrifugation at 7700g (9081.99rpm) twice with potassium phosphate buffer, pH 7.4 (KPB)
*Combine sample with 50μL 20μM HS-DNA  
*Combine sample with 50μL 20μM HS-DNA  
*Vortex for 1 hour
*Sonicate for 1 hour
*Rinse three times with KPB
*Rinse by centrifugation at 7700g (9081.99rpm) three times with KPB
*Re-suspend in 150μL KPB
*Re-suspend in 150μL KPB
Since times for centrifuging were not specified, 5 minute intervals will be done to determine appropriate length of time.<br.>
Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.>
Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.>


Latest revision as of 22:45, 26 September 2017

Silica-coating Reaction Main project page
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June 10, 2013

  • Silica-coating procedure from 05/24/2013 performed<br.>
    • MHA attachment performed, silica-coating reaction to be performed tomorrow
  • Sodium phosphate buffer, pH 9, made

Buffer Preparation and Calculations

Sodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO2 procedure.<br.> monosodium phosphate- MW: 119.98g/mol<br.> disodium phosphate- MW: 141.96g/mol, pKA: 6.82<br.> buffer- pH 9,concentration 10mM<br.> <br.> 9=6.82+log([base]/[acid])<br.> 151.36=(10-[base])/([acid])<br.> [acid](151.36+1)=10<br.> [acid]=0.066mM<br.> [base]=9.935mM<br.> <br.> Use 0.0094g NaH2PO4 and 1.19188g Na2HPO4 in 1L water.<br.>

New: Modifying Silica-Coated AuNPs with HS-DNA Procedure

adapted from Silica-Coated, Au/Ag Striped Nanowires for Bioanalysis

Reactions

Fuctionalizing the AuNPs@SiO2 with NH2

AuNPs@SiO2(aq*)+H2N(CH2)3Si(OCH3)3(l)→AuNPs@SiO2-NH2(aq*)<br.> '*'denotes colloidal system

Activating the NH2 Functionalized AuNPs@SiO2 with Sulfo-SMCC

AuNPs@SiO2-NH2(aq*)+C16H17N2O9SNa(aq**)→SSMCC-Activated AuNPs@SiO2-NH2(aq*)<br.> '*'denotes colloidal system<br.> '**"denotes dissolution in buffer, not water

Attaching the Thiolated DNA

SSMCC-Activated AuNPs@SiO2-NH2(aq*)+HS-DNA(aq)→Crosslinked S-DNA and AuNPs@SiO2-NH(aq*)<br.> '*'denotes colloidal system<br.>

Chemicals Needed and Safety

  • Ethanol
  • 15μL APS ((3-aminopropyl)trimethoxysilane)
  • 1mg SSMCC (sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane-1-carboxylate)
  • Potassium phosphate buffer, pH 7.4
  • Sodium phosphate buffer, pH 9
  • 150μL Silica-coated AuNPs
  • 50μL of 20μM HS-DNA

Procedure

  • Combine 150μL AuNPs@SiO2, 15μL APS, and 235μL ethanol
  • Sonicate solution for 30 minutes
  • Rinse sample three times by centrifugation at 7700g (9081.99rpm) with ethanol and two times with sodium phosphate buffer, pH 9 (SPB)
  • Make a separate solution of 1 mg sulfo-SMCC in 400μL of SPB
  • Add SSMCC solution to AuNP solution
  • Sonicate for 1 hour
  • Rinse by centrifugation at 7700g (9081.99rpm) twice with SPB
  • Rinse by centrifugation at 7700g (9081.99rpm) twice with potassium phosphate buffer, pH 7.4 (KPB)
  • Combine sample with 50μL 20μM HS-DNA
  • Sonicate for 1 hour
  • Rinse by centrifugation at 7700g (9081.99rpm) three times with KPB
  • Re-suspend in 150μL KPB

Since times for centrifuging were not specified, 5 minute intervals will be done to determine appropriate length of time.<br.> Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.>



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