RNase treatment of DNA

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[править] Protocol

  • prepare 37°C water bath in foam plastic box
  1. estimate DNA quality on 1.5% agarose gel (load ~0.2-0.6µg DNA);
  2. prepare reaction (up to 20% of RNA):
    DNA, /
    10x RNase ONE buffer
    RNase ONE, 10u/µl
    H2O
  3. 37°C, 1h;
  4. check presence of RNA on 1.5% agarose gel, store reaction in ice during electrophoresis (load ~0.2-0.6µg DNA; it is desirable to run control(untreated) DNA on the same gel);
  5. DNA purification
    • >20µg: Phenol/Chloroform extraction
    • 10-20µg: QIAprep column
    • <10µg: QIAquick column
          5xV of PB:
  6. snap freeze in liquid nitrogen and store at -20°C;


[править] Notes

  • Small RNA fragments are hardly visible on agarose gel, but their molar quantity may be high and they may inhibit polynucleotide kinase and polymerases.
  • Store RNase(s) in a special box in a freezer where there are no RNA-work related tubes.
  • It is possible to selectively remove single-stranded RNA and purify only double-stranded DNA on a silica column (protocol in Russian).
  • It is possible to shear DNA by ultrasound directly after RNase treatment w/o purification.
  • It is possible (but not desirable) to snap freeze DNA in liquid nitrogen and store at -20°C after RNase treatment w/o purification.


[править] Check list





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