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

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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. 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.