User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/04

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FCS Measurements and ZnPPIX Sample Prep Main project page
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June 4, 2013

FCS Measurements

Sonicate 10x diluted microspheres, dilute solutions 100x
(1/10)V=(1/100)(30μL)
V=3μL 10x diluted solution in 27μL distilled H2O

  • Dark red fluorescent microspheres, 0.2μm, blue solution, 660 excitation and 680emission, diluted 100x, 1 and 0.6 filters used
    • <N>=24
    • τ1=0.3068ms
    • <C>=40nM
  • Orange fluorescent microshperes, 0.1μm, pink solution, 540 excitation and 560 emission, diluted 100x, 1 and 0.6 and 0.4 filters used
    • <N>=813
    • τ1=121.465ms
    • <C>=1350nM
  • '#1': 1nM AuNP with MB and DNA
    • <N>=350
    • τ1=18.2086ms
    • <C>=581nM
  • '#2': 500pM AuNP with MB and DNA
    • <N>=302
    • τ1=5.6247ms
    • <C>=500nM
  • '#3': 100pM AuNP with MB and DNA
    • <N>=2.8/0.18/0.016
    • τ1=0.0033/0.00006/0.00002ms
    • <C>=4.6/0.30/0.027nM
  • '#4': 140.33nM MB with DNA
    • <N>=1120
    • τ1=4.0469ms
    • <C>=1870nM
  • '#5': 70.165nM MB with DNA
    • <N>=630
    • τ1=4.2336ms
    • <C>=1050nM
  • '#6': 14.033nM MB with DNA
    • <N>=116
    • τ1=4.2980ms
    • <C>=193n<
  • '#7': 140.33nM MB
    • <N>=480
    • τ1=2.3957ms
    • <C>=790nM
  • '#8': 70.165nM MB
    • <N>=55.2
    • τ1=33.8102ms
    • <C>=91.7nM
  • '#9': 14.033nM MB
    • <N>=100
    • τ1=0.0062ms
    • <C>=170nM

Notes

All samples prepared 06/03/2013, in addition to two modified microsphere solutions from Dr. Miller, were run on the FCS. Data was analyzed using N values, τ values, and C values were recorded. Tomorrow we will look in more detail at the time traces recorded directly from the instrument to look at differences in diffusion times.

Zinc Protoporphyrin Sample Prep: Calculations

Zinc protoporphyrin (ZnPPIX) samples will be prepared at the same concentrations, following the same procedure as the ThT samples.

  • 2.22μM ZnPPIX and 1.11265μM DNA

(5μM)V=(4.44μM)(300μL)
V=266.4μL 5μM ZnPPIX in 33.6μL
(53.1μ)V=(2.2253μM)(300μL)
V=12.57μL DNA in 287.43μL

  • ZnPPIX, DNA and AuNP

2[ThT]:1[DNA]→ 2.068μM ZnPPIX
10% of 250μL=25μL (within 10% allows for accurate concentration comparison)
Total solution volume: 275μL
(2.068×10-6M)(275×10-6L)=5.31476×10-10moles ZnPPIX
(5.31476×10-10moles)/(25×10-6L)=21.3μM ZnPPIX

Make 21.3μM ZnPPIX:
(5000μM)V=(21.3μM)(25μL)
V=0.107μL in 24.9μL
Add 25μL of 21.3μM ZnPPIX to 250μL DNA/AuNP solution

Concentrations:
DNA- 0.94μM
AuNP- 12.55nM
ZnPPIX- 1.9364μM

  • 2.22μM ZnPPIX

(5μM)V=(2.22μM)(300μL)
V=133.2μL ZnPPIX in 166.8μL

Notes

Samples will be prepared and spectra taken tomorrow.

Modifying Silica Coated AuNPs with HS-DNA Procedure- Changed(?)

  • Combine 150μL AuNPs@SiO2, 15μL APS and 235μL ethanol
  • Vortex solution for 30 minutes
  • Rinse sample three times by centrifugation at 7700g with ethanol and two times with KP buffer
  • Make a separate solution of 1 mg sulfo-SMCC in 400μL of KP buffer
  • Add SSMCC solution to AuNP solution
  • Vortex for 1 hour
  • Rinse twice with KP buffer
  • Rinse twice with PBS buffer
  • Combine sample with 50μL 20μM HS-DNA
  • Vortex for 1 hour
  • Rinse three times with PBS buffer
  • Re-suspend in 150μL PBS buffer


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