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

From OpenWetWare
Jump to navigationJump to search
Line 15: Line 15:
*Add 400μL of Zyppy Wash Buffer to the column. Centrifuge at top speed for 30 seconds.
*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.
*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.




Revision as of 14:08, 25 October 2013



 		<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2013/10/21&oldid=745775"