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

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(New: Modifying Silica-Coated AuNPs with HS-DNA Procedure)
Current revision (16:19, 20 June 2013) (view source)
(Procedure)
 
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*Silica-coating procedure from [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013] performed<br.>
*Silica-coating procedure from [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013] performed<br.>
**MHA attachment performed, silica-coating reaction to be performed tomorrow
**MHA attachment performed, silica-coating reaction to be performed tomorrow
 +
*Sodium phosphate buffer, pH 9, made
===Buffer Preparation and Calculations===
===Buffer Preparation and Calculations===
Sodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO<sub>2</sub> procedure.<br.>
Sodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO<sub>2</sub> procedure.<br.>
Line 23: Line 24:
Use 0.0094g NaH<sub>2</sub>PO<sub>4</sub> and 1.19188g Na<sub>2</sub>HPO<sub>4</sub> in 1L water.<br.>
Use 0.0094g NaH<sub>2</sub>PO<sub>4</sub> and 1.19188g Na<sub>2</sub>HPO<sub>4</sub> in 1L water.<br.>
===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure===
===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure===
 +
adapted from [http://pubs.acs.org/doi/full/10.1021/la7019846 Silica-Coated, Au/Ag Striped Nanowires for Bioanalysis]
====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<br.>
 +
'**"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 S-DNA and AuNPs@SiO<sub>2</sub>-NH<sub>(aq*)</sub><br.>
 +
'*'denotes colloidal system<br.>
 +
====Chemicals Needed and Safety====
====Chemicals Needed and Safety====
 +
*Ethanol
 +
*15μL APS ((3-aminopropyl)trimethoxysilane)
 +
*1mg SSMCC (sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane-1-carboxylate)
 +
**Must be stored at -20°C
 +
**Moisture sensitive, store in desiccant
 +
**[http://www.piercenet.com/instructions/2160581.pdf Manufacturer Instructions]
 +
*Potassium phosphate buffer, pH 7.4
 +
*Sodium phosphate buffer, pH 9
 +
*150μL Silica-coated AuNPs
 +
*50μL of 20μM HS-DNA
 +
====Procedure====
====Procedure====
-
*Combine 150μL AuNPs@SiO<sub>2</sub> (prepared 06/10/2013), 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 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
-
====Calculations====
+
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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Current revision

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

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*)
'*'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 S-DNA and AuNPs@SiO2-NH(aq*)
'*'denotes colloidal system

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.
Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.



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