Haynes Lab:Notebook/Engineering PC-TFs/2012/04/10

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4/11/12

Miniprep for flyPCD set

  • Pellet 1 - 5 mL bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature
  • Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to microcentrifuge tube
  • Add 250 µl Buffer P2 and mix by inverting tube 6 times until solution is clear.
  • Add 350 µl Buffer N3 and mix by inverting the tube 6 times.
  • Centrifuge for 10 minutes at 13,000 rpm
  • Apply the supernatant liquid to the QIAprep spin by decanting. Centrifuge for 30-60 second and discard the flow-through.
  • Wash the QIAprep column by adding .75 ml Buffer PE
  • Centrifuge for 60 seconds and discard the flow through.
  • Centrifuge for 1 min to remove residual wash buffer.
  • Place the QIAprep column in a clean 1.5 ml tube. To remove the DNA, add water to the center of the QIA spin columns, and centrifuge for 2 min.