User:Allison K. Alix/Notebook/Thesis Research/2013/01/29: Difference between revisions
No edit summary |
|||
(19 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|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> | ||
|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> </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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
|- | |- | ||
Line 9: | Line 9: | ||
-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 | |||
==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) | |||
Revision as of 17:33, 4 February 2013
ZnPPIX/DNA and ThT/DNA Sample Prep | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
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)
|