User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/25

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June 25, 2013

FCS Measurements

  • Samples prepared to be taken up to run on the FCS:
    • Antibodies with Alexa 532
    • Green fluorescent beads diluted 100x
    • Green fluorescent beads diluted 1000x
    • 100nM DNA/120nM MB
    • 1nM DNA/1.2nM MB
    • 500pM DNA/600pM MB
    • 100pM DNA/120pM MB
    • 500pM Oligo D fluorescent dye
    • 100pM Oligo D fluorescent dye


Oligo D Fluorescent Dye

Calibration Graph

Varied Concentrations of DNA and MB
Image:FCS data 2013_0625 DNA-MB comparison graphs.PNG

Calibration Graph
Image:FCS data 2013_0625 DNA-MB calibration.png


From the calibration graph for the Oligo D, there is a concentration drop-off between 50 and 100pM. More samples between this interval could be made to determine at what exact concentration the limit occurs. Otherwise, the trend between concentration of Oligo D and 1/<N> value is linear. The graphs were consistent with the exception of the 50pM sample, which could be due to the concentration drop-off observed in the calibration graph.
From the calibration graph for the DNA/MB samples, a concentration limit was not reached and the trend was linear. This means samples can be further diluted and run. The graphs were consistent with the exception of the final sample, 100pM DNA/120pM MB, which was expected to have a higher y-intercept value. This may be a concentration limit similar to the Oligo D, but was not evident in the <N> value. Samples will be made and run again tomorrow.

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