Monday, August 16, 2010

Purification of DNA and RNA from the same sample using the MasterPure Complete Kit

Many customers have asked how they can process both DNA and RNA from the same sample, using the MasterPure™ Complete DNA and RNA Purification Kit. The following protocol has been validated, and successfully used by customers:
  1. Follow the protocol for total nucleic acids (TNA) in the product literature, including Lysis (Part A) and the first two steps for Precipitation (Part B, steps 1 and 2).
At this point, split the supernatant into two equal portions. Label one tube “DNA” and the other “RNA”.
  1. Add 250 µl of isopropanol to both tubes, invert 30-40 times to mix.
  2. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
  3. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
  4. Rinse with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove residual ethanol with a pipet.
DNA Protocol (for the tube marked “DNA”)
  1. Resuspend the pellet in 100 µl TE Buffer.
  2. Add 1 µl of RNase A to sample and mix well.
  3. Incubate @ 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be needed.
  4. Add 100 µl of 2X T&C Lysis Solution, and mix by vortexing for 5 seconds.
  5. Add 100 µl of MPC protein precipitation reagent, mix by vortexing for 10 seconds. Place on ice for 3-5 minutes.
  6. Centrifuge for 10 minutes at ≥10,000 x g.
  7. Transfer the supernatant to a new microcentrifuge tube and discard the pellet.
  8. Add 250 µl of isopropanol. Invert 30-40 times to mix.
  9. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
  10. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
  11. Rinse twice with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove all residual ethanol with a pipet.
  12. Resuspend the DNA in 10-35 µl of TE Buffer.
RNA Protocol (for the tube marked “RNA”)
  1. Prepare 100 µl of DNase I solution by diluting 2.5 µl of RNase-Free DNase I up to 100 µl with 1X DNase buffer.
  2. Resuspend the pellet in 100 µl of DNase I solution.
  3. Continue with steps 3 through 12 from the DNA Protocol (above).

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