NA purification
Материал из Zbio
Содержание |
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
advantages disadvantages - 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
advantages disadvantages - 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
advantages disadvantages - 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)