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Latest revision as of 07:06, 20 November 2015

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Miniprep

Isolation of plasmid DNA from bacteria modified after Roti-Prep Plasmid MINI

material: Kit , aqua dest.
duration: 45 min

  1. resuspend bacteria in 250 µL resuspension buffer
  2. add 250 µL lysis buffer and mix gently
  3. incubate for 5 min at RT
  4. add 350 µL neutralisation buffer, mix gently
  5. centrifuge 10min, full speed, RT (big bench centrifuge)
  6. transfer supernatant to column
  7. centrifuge 30 sec, 10000g, RT (discard flow-through)
  8. add 750 µL wash buffer
  9. centrifuge 30 sec, 10000g, RT (discard flow-through)
  10. centrifuge 60 sec, 10000g, RT
  11. transfer column into 1.5 mL Eppendorff tube
  12. add 50 µL aqua dest (vary volume according to concentration needs, for higher yield the water may be prewarmed)
  13. incubate 5 min at RT
  14. centrifuge 1 min 10000 g RT

⇒ DNA is in the Eppendorff tube