Team:Mississippi State/Isolated Vector

From 2008.igem.org

  1. After centrifuge, empty supernatant into flask.
  2. Resuspend pellet in 250ul pl buffer.
  3. Vortex until cell pellet dissolves(looks milky.)
  4. Add 250ul p2 buffer, mix thoroughly by INVERTING 4-6 TIMES!!, don't vortex.
  5. Immediately add 350ul N3 buffer, mix immediately by inverting 4-6 times, mix thoroughly.
  6. Centrifuge 10min @ 13000rpm (17900g.)
  7. Get 3 spin columns from kit.
  8. Put supernatant into spin column via pipette.
  9. Centrifuge 30-60s, discard flow-through.
  10. Wash spin column, add 0.5ml PB buffer and centrifuge 30-60s, discard flow-through.
  11. Wash spin column, add .75ml PE buffer, centrifuge 60s.
  12. Discard flow-through, centrifuge additional 1min to remove residual wash buffer.
  13. place QIAprep column in clean 1.5ml centrifuge tube. To elate DNA, add 50ul water to the center of each QIAprep spin column, let stand for 5-10min, centrifuge 1min.
  14. Run Gel.
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