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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:
731nM(V)=6.49nM(1000μL)
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 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
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
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