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

FCS Measurements and ZnPPIX Sample Prep

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June 4, 2013 FCS Measurements Sonicate 10x diluted microspheres, dilute solutions 100x<br.> (1/10)V=(1/100)(30μL)<br.> 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)<br.> V=266.4μL 5μM ZnPPIX in 33.6μL<br.> (53.1μ)V=(2.2253μM)(300μL)<br.> V=12.57μL DNA in 287.43μL<br.> ZnPPIX, DNA and AuNP 2[ThT]:1[DNA]→ 2.068μM ZnPPIX<br.> 10% of 250μL=25μL (within 10% allows for accurate concentration comparison)<br.> Total solution volume: 275μL<br.> (2.068×10-6M)(275×10-6L)=5.31476×10-10moles ZnPPIX<br.> (5.31476×10-10moles)/(25×10-6L)=21.3μM ZnPPIX<br.> <br.> Make 21.3μM ZnPPIX:<br.> (5000μM)V=(21.3μM)(25μL)<br.> V=0.107μL in 24.9μL<br.> Add 25μL of 21.3μM ZnPPIX to 250μL DNA/AuNP solution<br.> <br.> Concentrations:<br.> DNA- 0.94μM<br.> AuNP- 12.55nM<br.> ZnPPIX- 1.9364μM<br.> 2.22μM ZnPPIX (5μM)V=(2.22μM)(300μL)<br.> V=133.2μL ZnPPIX in 166.8μL<br.> 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