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NB! pre-PCR operation

  1. attach vinyl tube to the nebulizer;
  2. dilute 1ng-10µg of genomic DNA in 50µl of TE;
  3. add Nebulization Buffer up to 700µl of, mix and put in the nebulizer;
  4. chill nebulizer in ice: ~10 min;
  5. connect air through the 0.2µm filter unit;
  6. work under fume hood!
  7. shear DNA (in ice) for 6min at 32-35psi (2,2-2,4 bar);
  8. collect DNA by 1 min. centrifugation at 4°C ~500g (1400rpm Beckman J6-HC) / should be ~400-600µl;
  9. snap freeze in liquid nitrogen, store at -20°C;

[править] Notes

  • Nebulization buffer: 50% Glycerol in TE (ρ~1.1g/ml)
  • ~30-50% of sample volume disappears as a fog
  • mean size of obtained fragments is ~300-400bp. It is unpractical to increase shearing time, it does not decrease the size of fragments, but more liquid is lost.
  • in principle, it is possible to reuse nebulizer for some applications. Wash it by several cycles of nebulizing pure water.
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