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

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(Chemicals Needed and Safety)
(Reactions)
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===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure===
===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure===
====Reactions====
====Reactions====
 +
=====Fuctionalizing the AuNPs@SiO<sub>2</sub> with NH<sub>2</sub>=====
 +
AuNPs@SiO<sub>2</sub><sub>(aq*)</sub>+H<sub>2</sub>N(CH<sub>2</sub>)<sub>3</sub>Si(OCH<sub>3</sub>)<sub>3</sub><sub>(l)</sub>→AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub><br.>
 +
'*'denotes colloidal system
 +
=====Activating the NH<sub>2</sub> Functionalized AuNPs@SiO<sub>2</sub> with Sulfo-SMCC=====
 +
AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub>+C<sub>16</sub>H<sub>17</sub>N<sub>2</sub>O<sub>9</sub>SNa<sub>(aq**)</sub>→SSMCC-Activated AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub><br.>
 +
'*'denotes colloidal system
 +
'**"denotes dissolution in buffer, not water
 +
=====Attaching the Thiolated DNA=====
 +
SSMCC-Activated AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub>+HS-DNA<sub>(aq)</sub>→Crosslinked thiolated DNA and AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub><br.>
 +
====Chemicals Needed and Safety====
====Chemicals Needed and Safety====
*Ethanol
*Ethanol

Revision as of 14:40, 10 June 2013

Silica-coating Reaction Main project page
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June 10, 2013

  • Silica-coating procedure from 05/24/2013 performed
    • 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.
monosodium phosphate- MW: 119.98g/mol
disodium phosphate- MW: 141.96g/mol, pKA: 6.82
buffer- pH 9,concentration 10mM

9=6.82+log([base]/[acid])
151.36=(10-[base])/([acid])
[acid](151.36+1)=10
[acid]=0.066mM
[base]=9.935mM

Use 0.0094g NaH2PO4 and 1.19188g Na2HPO4 in 1L water.

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

Reactions

Fuctionalizing the AuNPs@SiO2 with NH2

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

Activating the NH2 Functionalized AuNPs@SiO2 with Sulfo-SMCC

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

Attaching the Thiolated DNA

SSMCC-Activated AuNPs@SiO2-NH2(aq*)+HS-DNA(aq)→Crosslinked thiolated DNA and AuNPs@SiO2-NH2(aq*)

Chemicals Needed and Safety

  • Ethanol
  • 15μL, 1mM 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 (prepared 06/10/2013), 15μL APS and 235μL ethanol
  • Vortex solution for 30 minutes
  • Rinse sample three times by centrifugation at 7700g 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
  • Vortex for 1 hour
  • Rinse twice with SPB
  • Rinse twice with potassium phosphate buffer, pH 7.4 (KPB)
  • Combine sample with 50μL 20μM HS-DNA
  • Vortex for 1 hour
  • Rinse three times with KPB
  • Re-suspend in 150μL KPB

Calculations


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