User:Allison K. Alix/Notebook/Thesis Research/2013/01/29: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> ZnPPIX/DNA and ThT/DNA Sample Prep</span> | ||
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'''ZnPPIX:''' | '''ZnPPIX:''' | ||
5μM ZnPPIX already prepared | |||
-5μM ZnPPIX already prepared | |||
1. Prepare 1 μM 4 telomeric repeat DNA: | 1. Prepare 1 μM 4 telomeric repeat DNA: | ||
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x = '''2.656 μL''' in 97.344 μL H<sub>2</sub>O | x = '''2.656 μL''' in 97.344 μL H<sub>2</sub>O | ||
2. Prepare 1 μM 8 telomeric repeat DNA | 2. Prepare 1 μM 8 telomeric repeat DNA | ||
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x = '''1.428 μL''' in 98.572 μL H<sub>2</sub>O | x = '''1.428 μL''' in 98.572 μL H<sub>2</sub>O | ||
'''ThT''' | 3) Mix 100μL ZnPPIx with 100μL of each DNA sequences | ||
'''ThT:''' | |||
1. prepare 5mM tris-HCl buffer solution | 1. prepare 5mM tris-HCl buffer solution | ||
121.4 g/mol x 5mmol/L x 1mol/1000mmol x 50 mL x 1L/1000mL = '''0.030285 g''' tris in 50 mL H<sub>2</sub>O | |||
- Add 1 N HCl (~8 drops) to obtain pH ~ 7.25 | |||
2. prepare 3.5 μM ThT | |||
318.86 g/mol x 3.5mmol/L x 1mol/1000mmol x 10mL x 1L/1000mL = '''0.0111601 g''' in 10mL H<sub>2</sub>O -> 3.5mM | |||
(3.5 x 10^-3 M) (x) = (3.5 x 10^-6 M) (10mL) | |||
x = '''0.01mL''' in 9.99mL H<sub>2</sub>O | |||
3. Prepare 3.5μM, 3μM, 2.5μM, 2.0μM, and 1.5μM solutions of 4 telomeric repeat DNA | |||
a) (265.6μM)(x) = (3.5μM) (250μL) | |||
::x= '''3.29μL''' in 246.71μL tris-HCl buffer | |||
b) (265.6μM)(x) = (3.0μM) (250μL) | |||
::x= '''2.82μL''' in 247.18μL tris-HCl buffer | |||
c) (265.6μM)(x) = (2.5μM) (250μL) | |||
::x= '''2.35μL''' in 247.65μL tris-HCl buffer | |||
d) (265.6μM)(x) = (2.0μM) (250μL) | |||
::x= '''1.88μL''' in 248.12μL tris-HCl buffer | |||
e) (265.6μM)(x) = (1.5μM) (250μL) | |||
::x= '''1.41μL''' in 248.59μL tris-HCl buffer | |||
4. Prepare 3.5μM, 3μM, 2.5μM, 2.0μM, and 1.5μM solutions of 8 telomeric repeat DNA | |||
a) (142.8μM) (x) = (3.5μM)(250μL) | |||
::x= '''6.13μL''' in 243.87μL tris-HCl buffer | |||
b) (142.8μM) (x) = (3.0μM)(250μL) | |||
::x= '''5.25μL''' in 244.7μL tris-HCl buffer | |||
c) (142.8μM) (x) = (2.5μM)(250μL) | |||
::x= '''4.38μL''' in 245.6μL tris-HCl buffer | |||
d) (142.8μM) (x) = (2.0μM)(250μL) | |||
::x= '''3.50μL''' in 246.5μL tris-HCl buffer | |||
e) (142.8μM) (x) = (1.5μM)(250μL) | |||
::x= '''2.63μL''' in 247.4μL tris-HCl buffer | |||
5) Mix 100μL ThT with each of the prepared DNA samples in all concentrations | |||
'''For both ZnPPIX and ThT procedures:''' | |||
1) Heat all hybridization samples for ~20min at ~76°C | |||
2) Take absorbance and fluorescence of all (to be done next week) | |||
Revision as of 17:33, 4 February 2013
 ZnPPIX/DNA and ThT/DNA Sample Prep
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Objectives-Repeat ZnPPIX and DNA hybridizations in 5:1 ratios instead of 1:1 -Hybridize ThT and DNA strands ProceduresZnPPIX:
265.6μM (x) = 1μM (100μL) x = 2.656 μL in 97.344 μL H2O
142.8μM (x) = 1μM (100μL) x = 1.428 μL in 98.572 μL H2O 3) Mix 100μL ZnPPIx with 100μL of each DNA sequences
1. prepare 5mM tris-HCl buffer solution 121.4 g/mol x 5mmol/L x 1mol/1000mmol x 50 mL x 1L/1000mL = 0.030285 g tris in 50 mL H2O - Add 1 N HCl (~8 drops) to obtain pH ~ 7.25
318.86 g/mol x 3.5mmol/L x 1mol/1000mmol x 10mL x 1L/1000mL = 0.0111601 g in 10mL H2O -> 3.5mM (3.5 x 10^-3 M) (x) = (3.5 x 10^-6 M) (10mL) x = 0.01mL in 9.99mL H2O
a) (265.6μM)(x) = (3.5μM) (250μL)
b) (265.6μM)(x) = (3.0μM) (250μL)
c) (265.6μM)(x) = (2.5μM) (250μL)
d) (265.6μM)(x) = (2.0μM) (250μL)
e) (265.6μM)(x) = (1.5μM) (250μL)
a) (142.8μM) (x) = (3.5μM)(250μL)
b) (142.8μM) (x) = (3.0μM)(250μL)
c) (142.8μM) (x) = (2.5μM)(250μL)
d) (142.8μM) (x) = (2.0μM)(250μL)
e) (142.8μM) (x) = (1.5μM)(250μL)
5) Mix 100μL ThT with each of the prepared DNA samples in all concentrations
1) Heat all hybridization samples for ~20min at ~76°C 2) Take absorbance and fluorescence of all (to be done next week)
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