User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/01

FCS Measurements

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July 1, 2013 FCS Sample Preparation Green fluorescent beads 1000x diluted: from 10x dilution 10,000x diluted: from 10x dilution Oligo D: 20,000pM → 1000pM → 150pM 150pM: from 1000pM dilution 125pM: from 150pM dilution 100pM: from 150pM dilution 80pM: from 150pM dilution 60pM: from 150pM dilution 50pM: from 150pM dilution 40pM: from 150pM dilution 30pM: from 150pM dilution 20pM: from 150pM dilution 10pM: from 150pM dilution MB/DNA 1nM/800pM: from 2nM/1.6nM dilutions 900pM/720pM: from 50nM dilutions 800pM/640pM: from 50nM dilutions 700pM/560pM: from 2nM/1.6nM dilutions 600pM/480pM: from 2nM/1.6nM dilutions 500pM/400pM: from 2nM/1.6nM dilutions 400pM/320pM: from 2nM/1.6nM dilutions 300pM/240pM: from 2nM/1.6nM dilutions 200pM/160pM: from 2nM/1.6nM dilutions 100pM/80pM: from 2nM/1.6nM dilutions 75pM/60pM: from 2nM/1.6nM dilutions 50pM/40pM: from 2nM/1.6nM dilutions 25pM/12.5pM: from 2nM/1.6nM dilutions FCS Data: Calibration Curves <br.> Green fluorescent beads were run for 90s to align laser.<br.> Oligo D samples were run for 300s.<br.> MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.<br.> Notes Use green fluorescent beads to align laser Calibration curves did not turn out as expected (linear trend) DNA, not MB, should be in excess for DNA-MB samples to ensure all MB is bound Focus on three concentrations, run each sample several times, average curves based on best spectra taken today Oligo D: 150, 125, 100pM run three times at 500s per sample DNA/MB: 100pM DNA/80pM MB, 75/60, 50/40pM run three times at 500s per sample (increase to 700s per sample if spectra are noisey)