Team:LIKA-CESAR-Brasil/ExtractionProtocol

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   </head>
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Revision as of 02:02, 18 October 2014

LIKA | CESAR

NOTEBOOK

Extraction protocol of plasmid DNA (Miniprep)

For extraction of plasmid DNA was used Qiagen Spin Miniprep Kits.

Materials: Qiagen Miniprep Kit; Cell culture; 1.5 ml microtubes.

  • 1) Bacterial culture Centrifuge at 3000 g for 10 minutes.
  • 2) Discard supernatant containing sanitary water container.
  • 3) Resuspend pellet of bacterial cells in 250 ul of buffer P1. Transfer to a 1.5 ml tube.
  • 4) Add 250 uL of Buffer P2 and inverting the tube gently 4-6 times to mix. Not exceed five minutes with the lysis reaction.
  • 5) Add 350 ul of buffer N3 and invert the tube immediately and gently 4-6 times.
  • 6) Centrifuge for 10 minutes at 13,000 rpm in a microcentrifuge.
  • 7) Apply the supernatant from step 4 on QIAprep spin column by pipetting.
  • 8) Centrifuge for 30-60 s.
  • 9) Wash QIAprep spin column by adding 0.75 ul of buffer PE and centrifuged for 30-60 s. Remascente discard the liquid.
  • 10) Centrifuge for an additional 1 min to remove residual washing buffer.
  • 11) Place the QIAprep column in a clean 1.5 ml microfuge tube.
  • 12) For eluting the DNA, add 50 ul Buffer EB (10 mM Tris-HCl, pH 8.5) or water in the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
Marca LIKA Marca CESAR

Retrieved from "http://2014.igem.org/Team:LIKA-CESAR-Brasil/ExtractionProtocol"