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

2013 0703 10000x diluted fluor beads avg.PNG<br.> 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.<br.> <br.> 2013 0703 PBS avg.PNG<br.> 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.<br.> <br.> 2013 0703 DNA-MB avg.PNG<br.> 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.<br.>

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.<br.> 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. <br.> Green fluorescent beads are always run first to align laser for all following samples.<br.>