Haynes Lab:Notebook/Engineering PC-TFs/2013/10/21

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(October 21,2013)
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==October 21, 2013==
==October 21, 2013==
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'''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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<big>''Measure concentration of each fragment''</big>:
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* Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
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* Go to Nucleic Acid Quantification.
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* Pipette 2 µL of each DNA sequence into Take 3 plate.
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* 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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|-
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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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|-
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| CMV+MV9 (2)||1.92 ng/μL
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|-
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| CMV+MV9 (3)||27.394 ng/μL
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|}
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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


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