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