Team:LIKA-CESAR-Brasil/ExtractionRNA

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   </head>
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Revision as of 02:03, 18 October 2014

LIKA | CESAR

NOTEBOOK

Extraction of RNA

Materials needed Qiagen RNeasy mini kit

  • 1) Pipette 600 µl blood in a tube of 1.5 mL.
  • 2) Add 900 µl of lysis buffer, incubate for 10 minutes under stirring.
  • 3) After centrifugation for 5 minutes at 14.000rpm.
  • 4) Add to the pellet µl RLT buffer 600. Mix by pipetting.
  • 5) Pipette the lysate directly into a column of the RNeasy mini kit, centrifuge for 2 min at maximum speed.
  • 6) Add 1 volume of 70% ethanol to the homogenized lysate and mix well by pipetting. Do not centrifuge.
  • 7) Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently and centrifuge for 15 s in ≥8000 xg (≥10,000rpm).
  • 8) Add 500 µL of RPE buffer to the RNeasy spin column. Close the lid gently and centrifuge for 15 s in ≥8000 xg (≥10,000 rpm).
  • 9) Add 500 µL of RPE buffer to the RNeasy spin column. Close the lid gently and centrifuge for 2 minutes at ≥8000 xg (≥10,000 rpm).
  • 10) Place the RNeasy spin column in a new collection tube 1.5 ml (supplied). Add 30-50 µl RNAase free water directly onto the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥8000 xg (≥10,000 rpm) to elute the RNA.
Marca LIKA Marca CESAR

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