User:Klare Lazor/Notebook/Chem-496-001/2012/02/14

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Objective

  • Construct Concentration Curve of dye for fluorescence
  • Test Fluorescence of BSA and Dye

Description

Concentration Curve

  • Make a curve from a blank of buffer and water. Range of concentrations of dye up to 50µM
    • CALCULATION STOCK OF 50µM OF DYE in BUFFER and WATER
      • 515.3g/mol * 50E-6mols/L * 1L/1000 x 50mL = 0.001 grams of dye in 50mL Actual for both stocks 0.0015grams
    • CALCULATIONS FOR CONCENTRATION CURVE DYE + BUFFER FIRST
      • 50E-6mols/L * x = 10E-6 mols/L * 0.001L
        • Add 200µL of stock to 800µL of buffer
      • 50E-6mols/L *x = 5E-6 mols/L * 0.001 L
        • Add 100µL of stock to 900µL of buffer
      • 50E-6mols/L * x = 1E-6mols/L * 0.001 L
        • Add 20µL stock to 980 µL of buffer
      • 50E-6mols/L * x = 2E-6mols/L * .001L
  • REAL CONCENTRATIONS NEED TO BE CALCULATED FROM THE USE OF 0.0015 grams for the stock
    • TEMPORARY ESTIMATED CONCENTRATIONS
      • 1µM, 2µM, 5µM, and 10µM
      • BSA +DYE .... 30µL Dye+BSA and 1000µL of Buffer phosphate
      • To determine the amount of dye in Dye+BSA
        • (2.776mL)(15µM)=(x)(3.182mL [which is the mL of BSA and Buffer total for the synthesis])
        • x= 13µM for the synthesis solution of dye to bsa
        • 13µM(30/1030) = .381 µM of dye in the dye+BSA solution

NOTE: We will only make a concentration curve with the buffer and dye today

Data

Image:Bsa_and_dye.png

Figure1: Concentration curve and a spectra of the BSA + dye. The concentration of dye was determine to be 0.381µM. The addition of the dye to the BSA caused a shift 5nm confirming the attachment.

Notes

  • The dye+BSA produced an emittance of 525nm. The dye in solution produced an emittance 518nm. A total shift emittance was ≈ 7nm.


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