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

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(Objectives)
Current revision (19:33, 4 February 2013) (view source)
 
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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> ZnPPIX/DNA and ThT/DNA Sample Prep</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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-Repeat ZnPPIX and DNA hybridizations in 5:1 ratios instead of 1:1
-Repeat ZnPPIX and DNA hybridizations in 5:1 ratios instead of 1:1
-
- Hybridize ThT and DNA strands
+
-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 H<sub>2</sub>O
 +
 +
 +
2. Prepare 1 μM 8 telomeric repeat DNA
 +
 +
142.8μM (x) = 1μM (100μL)
 +
 +
x = '''1.428 μL''' in 98.572 μL H<sub>2</sub>O
 +
 +
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 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)

Current revision

ZnPPIX/DNA and ThT/DNA Sample Prep Main project page
Previous entry      Next entry

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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