NA purification

Материал из Zbio

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

• Phenol/Chloroform purification 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)