User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/01/29

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January 29, 2013

  • Concentration calculations-

1) make 100μL of 1μM 4 strand DNA:
2.656μL 4 strand DNA in 97.344μL filtered H2O
2) make 100μL of 1μM 8 strand DNA:
1.428μL 8 strand DNA in 98.527μL filtered H2O
3) make 6.49nM Molecular Beacon:
V=8.878μL MB in 991.12175μL unfiltered H2O
4) make TRIS buffer (pH of 7.2):
(121.14g/mol TRIS)(5mmol/L)(1mol/1000mmol)(50mL)(1L/1000mL)=0.030285g TRIS in 50mL solution

  • combine 0.0352g TRIS, 9 drops HCl, 5mL distilled H2O, 0.0025g TRIS, 20mL H2O
  • result: 5mM TRIS buffer solution with a pH of 7.2

5) make 3.5μM ThT
(318.86g/mol ThT)(3.5mmol/L)(1mol/10^3mmol)=1.116g/L(1L/1000mL)(10mL)=0.01116g ThT in 10mL solution

  • dissolve 0.0134g ThT (massed value, approximated for 0.01116g) in 10mL filtered H2O : 3.5mM ThT solution
  • dilute 10μL 3.5mM ThT solution in 9990μL filtered H2O : 3.5μM ThT solution

6) make DNA solutions at varying concentrations:
DNA (4 strand) concentration: 265.6μM
DNA (8 strand) concentration: 142.8μM

  • 1:265.6(V)=3.5(250μL)

V=3.29μL 4 strand DNA in 246.71μL TRIS buffer solution

  • 2:2.82μL 4 strand DNA in 247.18μL TRIS buffer solution
  • 3:2.35μL 4 strand DNA in 247.65μL TRIS buffer solution
  • 4:1.88μL 4 strand DNA in 248.12μL TRIS buffer solution
  • 5:1.41μL 4 strand DNA in 248.59μL TRIS buffer solution
  • 6:142.8(V)=3.5(250μL)

V=6.13μL 8 strand DNA in 243.87μL TRIS buffer solution

  • 7:5.25μL 8 strand DNA in 244.75μL TRIS buffer solution
  • 8:4.38μL 8 strand DNA in 245.62μL TRIS buffer solution
  • 9:3.50μL 8 strand DNA in 246.50μL TRIS buffer solution
  • 10:2.63μL 8 strand DNA in 247.38μL TRIS buffer solution

7)mix 100μL of each of the 10 DNA solutions with 100μL 3.5μM ThT