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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
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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