User:Allison K. Alix/Notebook/Thesis Research/2013/04/15: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav)
 
(5 intermediate revisions by one other user not shown)
Line 2: Line 2:
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
==Objectives==


* Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
* Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
Line 14: Line 14:
==Procedures==
==Procedures==


Part 1: Repeating Gel Electrophoresis
'''Part 1: Repeating Gel Electrophoresis'''


* Use same procedure as [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/11|04/11/2013]]
* Use same procedure as [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/11|04/11/2013]]
Line 20: Line 20:
* Prepare samples the same way
* Prepare samples the same way


Part 2: Reacting AuNP with DNA-thiol
'''Part 2: Reacting AuNP with DNA-thiol'''


1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/05|04/05/2013]] with 250μL 4% TEA containing 100mM DTT
1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/05|04/05/2013]] with 250μL 4% TEA containing 100mM DTT  
 
* New DNA concentration: 363.75nM, New ThT Concentration: 1.25μM


2. React 10 minutes
2. React 10 minutes
Line 28: Line 30:
3. Extract DTT using 4 2mL aliquots of ethyl acetate. DTT partitions into ethyl acetate and DNA partitions into aqueous phase
3. Extract DTT using 4 2mL aliquots of ethyl acetate. DTT partitions into ethyl acetate and DNA partitions into aqueous phase


4. Mix 232.4μL ThT/thiol-DNA with 232.4μL AuNP and 35.2μL sodium citrate buffer
4. Mix 232.4μL 1.25μM ThT/363.75nM thiol-DNA with 232.4μL 4.85nM AuNP and 35.2μL sodium citrate buffer
 
* Original concentration AuNP 19.4nM, concentration of solution needs to be 1:75 AuNP:DNA
 
363.75nM/74 = 4.85nM
 
19.4nM (x) = 4.85nM (250μL)
 
x = 62.5μL AuNP in 187.5μL flitered water
 
5. React for 10 minutes
 
6. Centrifuge 4 times, each time re-dispersing in 500 μL 50mM HEPES buffer
 
7. Save all supernatants and run Absorbance/Fluorescence Measurements on all


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Latest revision as of 22:38, 26 September 2017

Project name Main project page
Previous entry      Next entry

Objectives

  • Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
  • Bind AuNP to DNA-thiol hybrid

Procedures

Part 1: Repeating Gel Electrophoresis

  • Prepare samples the same way

Part 2: Reacting AuNP with DNA-thiol

1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized 04/05/2013 with 250μL 4% TEA containing 100mM DTT

  • New DNA concentration: 363.75nM, New ThT Concentration: 1.25μM

2. React 10 minutes

3. Extract DTT using 4 2mL aliquots of ethyl acetate. DTT partitions into ethyl acetate and DNA partitions into aqueous phase

4. Mix 232.4μL 1.25μM ThT/363.75nM thiol-DNA with 232.4μL 4.85nM AuNP and 35.2μL sodium citrate buffer

  • Original concentration AuNP 19.4nM, concentration of solution needs to be 1:75 AuNP:DNA

363.75nM/74 = 4.85nM

19.4nM (x) = 4.85nM (250μL)

x = 62.5μL AuNP in 187.5μL flitered water

5. React for 10 minutes

6. Centrifuge 4 times, each time re-dispersing in 500 μL 50mM HEPES buffer

7. Save all supernatants and run Absorbance/Fluorescence Measurements on all


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Allison_K._Alix/Notebook/Thesis_Research/2013/04/15&oldid=1012440"