DNase digestion

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NB! pre-PCR operation

  • DNase digestion should be done after polyA+ RNA purification[1]
  • preferably use Turbo DNase from Ambion[2]

prepare in advance:

» 37°C water bath

  1. for treatment of less than 2.5µg of RNA Turbo DNase should be diluted[3];
  2. combine all components in a 1.5mL LoBind tube and mix them

    Turbo DNase Buffer, 10x
    Turbo DNase

  3. incubate 30 min. at 37°C
  4. RNA cleanup (up to 40µg of RNA): RNeasy minElute[4];
  5. elute RNA in ??µl of ??
  6. snap freeze and store RNA:
    » -70°C for long-term storage
    » -20°C for work (~1 week)

[править] Notes

  1. otherwice ocasional RNA shearing would lead to under representation of 5' ends
  2. Turbo DNase from Ambion:
    • significantly more efficient for small concentration of DNA in solution, than normal DNase;
    • supplied in glycerol solution, may be stored at -20°C (e.g. DNase from Qiagen is supplied in solid form, should be diluted and stored at +4°C)
  3. if amount of DNA contamination in RNA sample is less than amount of RNA: use 0.2u of Turbo DNase for 1 µg of RNA
  4. in principle, it is possible to pause on this point: snap freeze and store the tubes at -20°C
Источник — «http://molbiol.ru/wiki/DNase_digestion»

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