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

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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

Image:FCS_data_2013_0701_OD,_MB-DNA_calibrations.PNG
Green fluorescent beads were run for 90s to align laser.
Oligo D samples were run for 300s.
MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.

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)


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