User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/03

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July 3, 2013

FCS Sample Prep

  • Green fluorescent beads
    • 10000x diluted, run for 90s
  • PBS
    • To check for background, run for 90s
  • DNA/MB
    • 100/80pM
    • 75/60pM
    • 50/40pM

FCS Data

Image:2013_0703_10000x_diluted_fluor_beads_avg.PNG
Green fluorescent beads diluted 10000x were run three times, 90s each; used to properly align laser for other samples. Laser not adjusted after bead samples were run.

Image:2013_0703_PBS_avg.PNG
PBS run twice, 90s each; to check for any possible background fluorescence. Average values show more fluorescence than expected, will be run again with next set of samples.

Image:2013_0703_DNA-MB_avg.PNG
Three different concentrations, run three times, 500s each. Samples were made using the same 1nM DNA and MB dilutions from 07/02/2013, allowed to hybridize at ~70C for 25 min then cooled for 20 min. 100pM DNA and 75pM DNA show expected trend: increase in y-intercept with decrease in concentration, but 50pM does not.

Notes

The inconsistency of the 50pM DNA/40pM MB sample has been observed twice in a row, which may indicate a lower detection limit around this concentration. However, since both sets of samples that have shown this inconsistency were made from the same dilutions (made on 07/02/2013), new samples will be completely remade to see if this trend continues. To look into this possibility further and for the construction of a calibration graph, 25pM DNA/20pM MB and 125pM DNA/100pM MB samples will be made for the next run.
PBS will also be run again, multiple times, for 200-300s, as the fluorescence and noise observed was significantly higher than expected. All samples have been diluted with PBS.
Green fluorescent beads are always run first to align laser for all following samples.



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