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.