Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2013/10/21"

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(October 21, 2013)
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|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
 
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
|style="background-color: #800000" 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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|style="background-color: #800000" 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}}}}]]}}
 
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==October 21, 2013==
 
==October 21, 2013==
'''Miniprep liquid cultures'''
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<big>''Miniprep liquid cultures''</big>
 
*Add 100 μL of 7X Lysis Buffer (Blue) to 600 μL of E.coli culture in a 1.5 ml microcentrifuge tube. Mix by inverting tube 4-6times.
 
*Add 100 μL of 7X Lysis Buffer (Blue) to 600 μL of E.coli culture in a 1.5 ml microcentrifuge tube. Mix by inverting tube 4-6times.
 
*Add 350 μL of cold Neutralization Buffer (Yellow), mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
 
*Add 350 μL of cold Neutralization Buffer (Yellow), mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
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Measure concentration of each fragment:
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<big>''Measure concentration of each fragment''</big>:
  
 
* Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
 
* Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
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* Place plate in EPOCH plate reader.
 
* Place plate in EPOCH plate reader.
  
 
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{| {{table}}
 
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| align="center" style="background:#efefef;"| '''Part'''
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| align="center" style="background:#efefef;"|'''Concentration'''
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| KAH126 (1)|| 22.239 ng/μL
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|-
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| KAH126 (2)||57.672 ng/μL
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|-
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| CMV+MV9 (1)||11.323 ng/μL
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| CMV+MV9 (2)||1.92 ng/μL
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| CMV+MV9 (3)||27.394 ng/μL
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|}
  
 
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Latest revision as of 22:30, 26 September 2017


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October 21, 2013

Miniprep liquid cultures

  • Add 100 μL of 7X Lysis Buffer (Blue) to 600 μL of E.coli culture in a 1.5 ml microcentrifuge tube. Mix by inverting tube 4-6times.
  • Add 350 μL of cold Neutralization Buffer (Yellow), mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
  • Centrifuge at 11,000-16,000 g for two minutes.
  • Transfer the supernatent into the Zymo-Spin Column. Place column in 2 ml collection tube and centrifuge for 15 seconds at top speed. Discard flow-through.
  • Add 200μL of Endo-Wash Buffer to the column. Centrifuge for 15 seconds at top speed.
  • Add 400μL of Zyppy Wash Buffer to the column. Centrifuge at top speed for 30 seconds.
  • Transfer column to 1.5 microcentrifuge tube and add 50μL of Zyppy Elution Buffer directly to column. Centrifuge for 1 minute at top speed to elute DNA.


Measure concentration of each fragment:

  • Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
  • Go to Nucleic Acid Quantification.
  • Pipette 2 µL of each DNA sequence into Take 3 plate.
  • Place plate in EPOCH plate reader.
Part Concentration
KAH126 (1) 22.239 ng/μL
KAH126 (2) 57.672 ng/μL
CMV+MV9 (1) 11.323 ng/μL
CMV+MV9 (2) 1.92 ng/μL
CMV+MV9 (3) 27.394 ng/μL