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

From OpenWetWare
Jump to navigationJump to search
Line 62: Line 62:
*Rinse three times with KPB
*Rinse three times with KPB
*Re-suspend in 150μL KPB
*Re-suspend in 150μL KPB
Concentrations may be doubled.<br.>
Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.>


====Calculations====
====Calculations====

Revision as of 12:40, 20 June 2013

Silica-coating Reaction <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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
  • Vortex 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
  • 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

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

Calculations


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/10&oldid=704278"