NA purification

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There is no "the best of the best" protocol. Different procedures have different advantages:

» best output: phenol/chloroform with ethanol precipitation; important for multistage procedures
» fast procedure: silica-spin columns



[править] Silica spin-purification

» output 80-95%
» purify most of enzymes w/o Proteinase K treatment
» large input volume: vacuum loading
advantagesdisadvantages
  • fast procedure
  • eluted DNA (in low-salt buffer) is ready for enzymatic reactions
  • safe and robust procedure: it is impossible to "lose a pellet"
  • it is possible to purify extremely low amounts of DNA w/o carriers
  • it is possible to isolate DNA from agarose gel
  • high output variability: 60-95%
  • mean output (~90%) is lower, than for phenol/chloroform with ethanol precipitation (>95%)
  • procedure slightly inhibit some enzymes
  • inaccurate washing may result in chaotropic salt contamination



[править] Ampure

» output >95% (normally, in 98-100% range)
» nice removal of short nucleic acids



[править] CTAB (cetyltrimethylammonium bromide)

» leaves neutral carbohydrates in solution. The method will obviously not remove anionic polysaccharides. NA purification from plants.
» Cetyltrimethylammonium is exchanged out be washing the pellet with an alcoholic salt.



[править] Phenol extraction and Ethanol precipitation

» output ~95% w/o phase lock, ~98% with phase lock
» phase lock increase the output and makes it more stable
» even for "pure DNA solutions" previous Phenol/Chloroform purification increase the output and make it more stable (surprisingly)
» "phase lock" and "precipitation carrier" make procedure about as hendy and fast as silica spin columns
» carriers: "Glycogen" and "GlycoBlue" — are practically the same results. We prefer "Glycogen" just because it is less modified product
» some enzymes require Proteinase K digestion
advantagesdisadvantages
  • high output (>95%)
  • very stable (especially with precipitation carrier)
  • purified DNA may be diluted in any desirable volume
  • it is possible to isolate DNA from LMP agarose
  • more difficult procedure, if compare with silica spin purification. Especially w/o "phase lock" and "precipitation carrier". Tricky phase separation, it is possible to "lose a pellet"
  • longer than silica spin purification. Especially w/o "phase lock" and "precipitation carrier".
  • it is impossible to purify extremely low amounts of DNA w/o carriers
  • bad smell



[править] Gel filtration spin columns

» output >95%
» only for low-molecular weight contaminations:
change buffer, pH, salt
dNTP's, NTP's, labeled nucleotides
small oligonucleotides
» be careful with solutions with detergent: relatively large miceles may transfer low-molucular weight substances through the column
advantagesdisadvantages
  • fast procedure
  • high output
  • eluted DNA may be in any desirable buffer
  • safe and robust procedure
  • eluted volume is higher, than input volume: ~40-50µl eluted if 30µl is loaded
  • only low-molecular weight contaminations
  • unclear results for medium-molecular weight contaminations(such as oligonucleotides)


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