Team:Hawaii/Protocols/gel extraction

From 2008.igem.org

Gel Extraction

  • Using Qiagen MiniElute Gel Extraction Kit

Protocol

  1. Cut band(s) from gel.
  2. Determine weight of gel.
    • If weight of gel >400mg, run as 2+ extractions in different tubes.
  3. Add Buffer QG.
    • For gels <2% agarose, add 3 gel volumes of QG.
    • For gels >2% agarose, add 6 gel volumes of QG.
  4. Incubate at 50C, shaking every 2-3 minutes, until gel is dissolved.
  5. Check the color of the solution is yellow. If the solution is orange, add 3M sodium acetate until solution is yellow (indicates neutral pH).
  6. Add 1 gel volume of isopropanol and mix by inverting.
  7. Transfer solution to elution spin column (purple).
  8. Centrifuge 1 min. at room temperature at 13,000 rpm.
  9. Discard flow through.
  10. Add 500 μl Buffer QG.
  11. Centrifuge 1 min. at room temperature at 13,000 rpm.
  12. Discard flow through.
  13. Add 750 μl Buffer PE and incubate 2 min.
  14. Centrifuge 1 min. at room temperature at 13,000 rpm.
  15. Discard flow through.
  16. Transfer spin column to fresh eppendorf tube.
  17. Add 10 μl Buffer EB and incubate 1 min.
    • Add buffer directly onto filter in the spin column.
  18. Centrifuge 1 min. at room temperature at 13,000 rpm.
  19. Collect flow through and store at -20C.

Notes

  • DNA recovery is ~80%.
Retrieved from "http://2008.igem.org/Team:Hawaii/Protocols/gel_extraction"