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.