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

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(Summary)
(September 7, 2013)
Line 11: Line 11:
 
*Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
 
*Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
 
*Grow the cultures for 10-12 hrs.
 
*Grow the cultures for 10-12 hrs.
 +
 +
[Image:CMV promoter9-6-2013|200px]
 +
 +
'''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 20:18, 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.

[Image:CMV promoter9-6-2013|200px]

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.