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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(June 10, 2013)
(Chemicals Needed and Safety)
Line 26: Line 26:
====Reactions====
====Reactions====
====Chemicals Needed and Safety====
====Chemicals Needed and Safety====
 +
*Ethanol
 +
*15μL, 1mM 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> (prepared 06/10/2013), 15μL APS and 235μL ethanol

Revision as of 13:02, 10 June 2013

Silica-coating Reaction Main project page
Previous entry      Next entry

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

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


Personal tools