Team:DTU-Denmark/Plasmid purification by miniprep

From 2011.igem.org

(Difference between revisions)
Lri (Talk | contribs)
(Created page with "{{:Team:DTU-Denmark/Templates/Standard_page_begin|Plasmid purification}} Plasmid purification by miniprep (Zymo Research Group) # Spin down 2 ml of cell culture in a 2 ml Eppend...")
Newer edit →

Revision as of 15:06, 18 September 2011

Plasmid purification

Plasmid purification by miniprep (Zymo Research Group)

  1. Spin down 2 ml of cell culture in a 2 ml Eppendorf tube at 11.000g for 5 min
  2. Remove supernatant, spin again down for 10 seconds, remove supernatant (if the pellet is not sufficient, repeat step 1 in the same tube)
  3. Resuspend your pellet in 600 ul TE buffer
  4. Add 100 µl 7X Lysis Buffer (blue) and mix by inverting the tube 4-6 times. Proceed to the next step within 2 minutes.
  5. Add 350 µl cold Neutralization Buffer (Yellow) and mix carefully. The sample will turn yellow and a yellowish precipitate will occur, then the reaction is finished. Invert the tube an additional 2-3 times to ensure complete neutralization.
  6. Centrifuge at 11,000 g for 5 min.
  7. Transfer the supernatant (ca. 875 µl) into a column. Avoid disturbing the cell debris pellet.
  8. Place the column into a collection tube and centrifuge for 30 seconds.
  9. Discard the flow-through and place the column back into the same collection tube.
  10. Add 200 µl Endo-Wash-Buffer to the column and centrifuge for 30 seconds.
  11. Add 400 µl of ZyppyWash Buffer to the column and centrifuge for 30 seconds.
  12. Transfer the column into a clean 1,5 ml Eppendorf tube and then add 50 µl Zyppy Elution Buffer directly to the column. Let the product(s) stand for 1-15 minutes on the table at RT.
  13. Centrifuge for 30 seconds to elute the plasmid DNA.


Retrieved from "http://2011.igem.org/Team:DTU-Denmark/Plasmid_purification_by_miniprep"