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

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(October 21,2013)
(October 21, 2013)
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*Add 400μL of Zyppy Wash Buffer to the column. Centrifuge at top speed for 30 seconds.
 
*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.
 
*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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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.
 +
  
  

Revision as of 14:08, 25 October 2013


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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.