User:Allison K. Alix/Notebook/Thesis Research/2013/01/29

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ZnPPIX/DNA and ThT/DNA Sample Prep Main project page
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Objectives

-Repeat ZnPPIX and DNA hybridizations in 5:1 ratios instead of 1:1

-Hybridize ThT and DNA strands

Procedures

ZnPPIX:


-5μM ZnPPIX already prepared


1. Prepare 1 μM 4 telomeric repeat DNA:

265.6μM (x) = 1μM (100μL)

x = 2.656 μL in 97.344 μL H2O


2. Prepare 1 μM 8 telomeric repeat DNA

142.8μM (x) = 1μM (100μL)

x = 1.428 μL in 98.572 μL H2O

3) Mix 100μL ZnPPIx with 100μL of each DNA sequences


ThT:

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


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 H2O -> 3.5mM

(3.5 x 10^-3 M) (x) = (3.5 x 10^-6 M) (10mL)

x = 0.01mL in 9.99mL H2O


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)



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