Ethanol precipitation

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[править] General protocol

  1. (optional) add precipitation carrier[1] and mix;
  2. add salt[2] and mix
    » AcNH4 ⇒ 2.0M
    » LiCl ⇒ 0.8M
    » NaCl ⇒ 0.2M
    » AcONa ⇒ 0.3M
  3. add either 2.5xV of Ethanol or 1xV of Isopropanol and mix[3];
  4. incubate from {NT, 5min} to {-20°C, ON} depending on DNA concentration;
  5. centrifugation from {14krpm, room temperature, 5min} to {27krpm, 4°C, 1-2h} depending on DNA concentration;
  6. 1-2x wash with 70% EtOH;
  7. dissolve NA in EB, TE, H2O or other aqueous solution

[править] Precipitation of large amounts of long genomic DNA

  1. add salt and mix
    » NaCl ⇒ 0.2M for precipitation from SDS-containing solution
    » AcNH4 ⇒ 2.0M — in other cases
  2. add either 2.5xV of Ethanol or 1xV of Isopropanol, NB! do not mix(!);
  3. incubate at room temperature for 5-20 min.;
  4. when DNA forms clog on the border of phases: mix tube content carefully;
  5. two times 70% EtOH washing:
    ○○   transfer DNA clog into the tube with 70% EtOH
    ○○   accurately mix and incubate for 3-5min.
  6. centrifugation 14krpm, room temperature, 5min;
  7. 1x wash pellet with 70% EtOH;
  8. remove all traces of 70% EtOH and slightly(!) dry the pellet[4];
  9. dissolve DNA in EB by rotation at 4°C for 20 min. - overnight

[править] Auto-calculation

  1. DNA sample:
        » volume [ml]:
        » ~amount [µg]:

  2. (optional) add and mix precipitation carrier: , :

  3. add and mix salt , :

  4. select and tube type: :

  5. incubate:
  6. centrifugation:
  7. 2x wash with 70% EtOH:

  8. dissolve NA in EB:
        » desirable final concentration [ng/µl]:
        » EB volume [µl]:

[править] Notes

  • Ethanol precipitation is a group related methods. All of them work fine. Usually, selection of the particular procedure (salt, cooling, centrifugation time, etc.) is a matter of traditions and prejudices.

  • The idea of the method: DNA (RNA) form aggregates in 70% EtOH in the presence of salt (aggregation accelerates at low temperature). Aggregates are pelleted by centrifugation. After precipitation cation of nucleic acid is that used for precipitation.

  • Some other substances are precipitates together with nucleic acids:
» polysaccharides (nice example: purification of RNA from liver. A lot of glycogen copurified with RNA.)
» phosphates >>1mM
» EDTA >10mM

[править] Precipitation carriers

  • are used for precipitation of small amounts of nucleic acids. Couple usefull effects:
» precipitation efficiency increases
» pellet is more visible, lower chances to loose it occasionally

  • all coprecipitants listed below
» remain in water phase after extraction of phenol or chloroform
» should be stored at -20°C

  • the main factor for selection of coprecipitant: it should not interfere with further application
coprecipitantstockadd tonotes
at least 10µg per tube
  • do not incubate NA solution with glycogen at low temperature: mix well and centrifuge 20 min. at room temperature
  • do not adsorb light in 260-280nm
linear polyacrylamide2.5µg/µl10-20µl/ml,
at least 10µg per tube
  • good coprecipitant for >20bp NA fragments
  • practically does not coprecipitate fragments <10bp
  • usefull for precipitation of labeled oligonucleotides
  • do not adsorb light in 260-280nm
at least 5µg per tube
  • NB! inhibitor of polynucleotid kinase, interfere with spectrophotometric measurement of concentration

[править] Salt/Ethanol recipies

    conc.stocksalt2.5xV EtOH1xV Isoprop

  • maximum sample volume for different types of tubes:
tube [ml]Etanol / Isopropanol
type [ml]max. vol. [ml]AcNH4, 10MLiCl, 7.5MAcONa, 3M
LiCl, 8M
NaCl, 5M
0.20.2761 / 108 µl69 / 120 µl70 / 121 µl74 / 130 µl
0.50.56128 / 224 µl142 / 250 µl144 / 252 µl153 / 270 µl
1.5340 / 600 µl380 / 670 µl386 / 675 µl410 / 720 µl
2.00.46 / 0.80 ml0.51 / 0.89 ml0.51 / 0.90 ml0.55 / 0.96 ml
153.4 / 6.0 ml3.8 / 6.7 ml3.9 / 6.8 ml4.1 / 7.2 ml
5011.4 / 20 ml12.8 / 22.3 ml12.9 / 22.5 ml13.7 / 24 ml

Ethanol or Isopropanol

  • substitution of Ethanol by Isopropanol has only one advantage: it is possible to use tubes with smaller volume, because 1xV of Isopropanol should be added instead of 2.5xV of Ethanol. The main disadvantage: Isopropanol is less volatile and 70% EtOH washing is obligatory.
  • Ethanol and Isopropanol should be stored in tightly(!!!) closed bottles/tubes, because any ethanol solutions stronger than 40% adsorbs water from the air. * it is convenient to store working aliquots in refrigerator.

Type and concentration of monovalent salt

saltstockadd tonotes
  • low coprecipitation of dNTPs (2x reprecipitation leads to removal of ~99% dNTPs)
  • low precipitation of polysaccharides
  • NB! NH4+ ions are inhibitors of polynucleotidkinase
  • good solubility in Ethanol solutions
  • LiCl (without EtOH) selectively precipitates long RNA
  • NB! Li+ ions are inhibitors of cell-free translation and reverse transcription
  • the best choice for solutions with SDS (other salts precipitates SDS much better, than NaCl)
NaAc, pH5.23M0.3M
  • routine precipitation of DNA
  • MgCl2, 10mM improves precipitation.

[править] Cooling and centrifugation

  • centrifugation time/force have more effect on precipitation than time/temperature of incubation before centrifugation
  • incubation:
» no incubation if glycogen is added as a carrier
» high NA concentrations >10µg/ml ⇒ room temperature, ~5min.
» middle concentrations 20ng/ml<NAconc<10µg/ml ⇒ -20°C, 15-30min.
» low concentration (<20ng/ml) or small size (<100bp) ⇒ -20°C, 1h - overnight

  • centrifugation:
» high NA concentrations >10µg/ml ⇒ 10-14krpm, room temperature, ~5min.
» middle concentrations 20ng/ml<NAconc<10µg/ml ⇒ 10-14krpm, room temperature - 4°C, 10-15min.
» low concentration (<20ng/ml) or small size (<100bp) ⇒ 14-27krpm, 4°C, 20min - 1h

  • often, NA pellet is invisible. It is necessary to know exactly on which side of the tube you should look for the pellet: mark this side by marker, place cap tails of 0.2-2.0ml tubes outside of the rotor.

[править] 70% EtOH washing

  • obligatory for Isopropanol pelleting
  • this operation removes original buffer, traces of organic solvents, etc. Normally, one wash is enough. Use two washes for:
» massive pellets
» precipitation of crude extracts (for example w/o phenol/chlorophorm extraction)
  • massive pellets: it is not enough just to wash pellet. It is necessary shake tube for ~1-2min. (would be nice, it pellet unstick from the wall)

  • common procedure after pelleting of NA:
» remove most of the supernatant: pour out or aspirate
» centrifugation: 14krpm, NT, 3min
» remove the rest of supernatant by aspiration
» add 70% EtOH and mix
» centrifugation: 14krpm, NT, 3min
» remove most of the supernatant: pour out or aspirate
» centrifugation: 14krpm, NT, 3min
» remove the rest of supernatant by aspiration

  • volumes of 70% EtOH for different types of tubes:
tube [ml]70% EtOH volume

[править] Drying and dissolving

  • do not overdry NA (drying under the vacuum is especially dangerous!)
» it is difficult to dissolve overdried DNA (especially long one)
» short DNA (<400bp) may denature unde the vacuum

  • common procedure to prevent overdrying:
» remove most of the 70% EtOH: pour out or aspirate
» centrifugation: 14krpm, NT, 3min
» remove the very rest of supernatant by aspiration

now DNA is redy to be solved. Traces of Ethanol do not inhibit any enzymatic reaction.

  • if you want to dry pellet:
» do it on room temperature
» (preferable) under fume hood — for soft steady stream of air
» continuously control drying: add buffer just after dissapearance of milky-white colour of the pellet

  • if the pellet does not want to dissolve:
» NB! do not try to solubilize pellet by pipetting: pellet may stick to the wall of the tip (and usually do it)
» rotate tube at 4°C for 20 min. - overnight
» heat tube to 70°C for 5min
» freeze at -20°C/thaw the tube couple of times

last two operations are distructive: may shear the long DNA (>3kbp)

  • it is more difficult to solve NA in buffer with high ionic strength. If NA should be dissolved in buffer with high ionic strength do it in two steps:
» dissolve NA in low-salt buffer
» increase ionic strength

  1. we use glycogen (20µg/µl): 0.5µl for up to 1ml of DNA solution
  2. we prefer NaOAc
  3. V — volume with salt
  4. pellet borders lose milky-white colour

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