RNeasy with DNase Treatment

 Dominik Haudenschild, March 2000 UC Davis
  1. Disrupt tissue and homogenize lysate
    use 600µl of buffer RLT per 30mg of tissue
    add 10µl ßMe per 1ml buffer RLT before use
  2. Centrifuge lysate for 3 min at max speed
  3. Add 1 volume 70% ethanol to cleared lysate and mix well
  4. Apply 700µl of sample to RNeasy column, centrifuge _10,000rpm
    discard flowthru and reuse collection tube in step 5
  5. Pipet 350µl buffer RW1 into the spin column, centrifuge _10,000rpm
    discard flowthru and reuse collection tube in step 6
  6. Add 10µl DNase-I stock solution to 70µl buffer RDD. Mix gently
    DNase-I is especially sensitive to physical denaturation. Mixing should only be
    carried out by gently inverting the tube. Do NOT vortex.
  7. Pipet the DNase-I incubation mix directly onto the spin-column membrane
    place on benchtop for 15 minutes. Make sure to pipet the DNase mixture directly
    onto the spin column membrane. Incomplete digestion will occur if mix sticks to
    walls of tube.
  8. Pipet 350µl Buffer RW1 into the spin column, centrifuge _10,000rpm.
     discard flowthru and collection tube
  9. Place spin column onto new collection tube, pipet 500µl buffer RPE, centrifuge
    discard flowthru and reuse collection tube in step 10
  10. Pipet 500µl buffer RPE into column, centrifuge maximum speed _2 minutes to
    dry membrane.
  11. Transfer to 1.5ml collection tube, elute with 30-50µl of water.