User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/02

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July 2, 2013

FCS Sample Prep

  • Green fluorescent beads
    • 10000x diluted
    • To be run 3x, 90s
  • Oligo D
    • 150, 125, 100pM
    • To be run 3x, 300s (increase to 500s if noisey)
  • DNA/MB
    • 100/80, 75/60, 50/40pM
    • Heated at ~70C for 25 min, cooled for at least 20 min
    • To be run 3x, 500s (increase to 700s if noisey)

FCS Data

Image:FCS_data_2013_0702_beads_curve.PNG
Beads used to align laser; three samples run at 90s each, average values used. Curve turned out as expected.
Image:FCS_data_2013_0702_Oligo_D_comparison.PNG
Oligo D: three samples run at each concentration, 500s each, average values used. Y-intercepts increase as concentration decreases, as expected.
Image:FCS_data_2013_0702_DNA-MB_comparison.PNG
DNA/MB: three samples run at each concentration, 500s each, average values used. Y-intercepts increase as concentration decreases until 50pM. DNA in excess rather than MB to ensure binding.
Image:FCS_data_2013_0702_calibration.PNG
Calibration graphs for Oligo D and DNA/MB samples. Oligo D trend turned out as expected; DNA/MB trend dropped at 50pM concentration. DNA/MB samples will be remade and run again tomorrow.


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