Haynes Lab:Notebook/Engineering PC-TFs/2013/09/07: Difference between revisions

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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>
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|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>}} <br> <font color="#FFCC00">'''Engineering PC-TFs'''
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==Summary==
==September 7, 2013==
*  
'''Grow liquid culture of CMV promoter'''
*Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp.
*Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
*Grow the cultures for 10-12 hrs.
 
[[Image:cmv.jpg|200px|CMV promoter]]
 
'''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.
 




Revision as of 20:29, 7 September 2013



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September 7, 2013

Grow liquid culture of CMV promoter

  • Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp.
  • Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
  • Grow the cultures for 10-12 hrs.

CMV promoter

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.



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