Phenol/Chloroform purification

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[править] DNA: Phase Lock Tubes (PL-tubes) protocol

    [править] sample preparation

  1. (optional) proteinase K digestion:
    » add to NA solution:
    total:
    NA sample 
    ProtK buffer, 10x1x
    H2OmQ
    SDS, 2%0.2%
    ProtK, 20µg/µl150µg/ml

    » mix, incubate 15 min at 37°C
  2. add salt to the sample (skip it, if proteinase K digestion was performed[1]):
    » NaCl to 0.2M or
    » AcONa to 0.3M

  3. [править] purification

    NB! see "Notes" about mixing and transfer of aqueous phase
  4. centrifuge ~0.5 min. PL-tube[2];
  5. transfer sample and 1xV Ph/Ch/I[3] to the PL-tube;
  6. shake the tube 2 min;
  7. centrifuge[4];
  8. add 1xV Ch/I[5];
  9. shake[6] the tube 2 min;
  10. centrifuge[4];
  11. transfer the upper phase to a new tube;

  12. [править] precipitation

  13. perform "ethanol precipitation"[7], NB! salt is already in the buffer:
    » add 2.5xV of 100% EtOH and mix
    » 15-30 min. at -20°C in cooling block
    » centrifugation: 14krpm, room temperature - 4°C, 10-15 min.
    » 2x wash with 70% EtOH
    » dilute in EB




[править] DNA: normal protocol

  1. sample preparation: as in the previous protocol;
  2. (optional) Phenol extraction:
    » add 1xV Ph[8] to the sample
    » shake[9] the tube 3 min
    » centrifuge[4] 5 min
  3. Phenol/Chloroform extraction:
    » transfer upper (aqueous) phase[10] into a new tube with 1xV Ph/Ch/I[3]
    » shake[9] the tube 3 min
    » centrifuge[4] 5 min
  4. Chloroform extraction:
    » transfer upper (aqueous) phase[10] into a new tube with 1xV Ch/I[5]
    » shake[9] the tube 3 min
    » centrifuge[4] 5 min
  5. transfer the upper phase to a new tube;
  6. ethanol precipitation: as in the previous protocol




[править] RNA

similar to DNA extraction. The only difference: using of acidic phenol, instead of neutral one. Both neutral and acidic phenols have the same efficiency in protein removal, but acidic also removes DNA from the solution.


[править] Solutions

[править] 10x ProtK buffer

store at +4°C

conc.stock50ml
NaCl2M5M20ml
Tris-HCl, pH8-8.2500mM1M25ml
EDTA50mM0.5M5ml



[править] Proteinase storage solution

store at -20°C

conc.stock50ml
CaCl21mM1M50µl
Tris-HCl, pH8-8.210mM1M500µl
Glycerol, ρ=1.25g/ml50%100%25ml / 31.25g /
H2O mQ24.45ml



[править] Proteinase K solution, 20µg/µl

» prepare ~20mg aliquote of solid Proteinase K in 2ml LoBind tube;
» weight it on analitical balance;
» add 50µl of Proteinase storage solution per each "mg" of Proteinase;
» solubilize Prot.K;
» dispense on ~100µl portions in signed 0.5ml LoBind tubes;
» store:
working aliquotes at -20°C
storage aliquotes at -70°C



[править] Acidic phenol buffer, 10x

store at 4°C

conc.stock100ml
AcONa, pH 5.10.5M3M16.7ml
EDTA100mM0.5M20ml
H2O mQ63.3ml

NB! dilute to 1x before use


[править] Phenol, acidic

store at 4°C in the dark (working aliquote), -20°C (storage for ~year)
NB! dangerous substance!

» check quality of phenol crystals: should be white; should not be any yelow of red colour
» add ~1/5x of V H2O (two phases: aqueous phase is ~0.1x of phenol)
» add and mix:
8-Hydroxyquinoline0.1% of phenol (~1g per liter)
βMeEtOH (2-Mercaptoethanol)0.02% of phenol (~200µl per liter)



NB! for RNA extraction acidic phenol should be equilibrated with ~0.1x V of 1x Acidic phenol buffer

» check pH of aqueous phase (pipette on the pH-strip), should be pH ~5
» if necessary, shift pH down by diluted (1:100) acetic acid



[править] Phenol, neutral

store at 4°C in the dark (~2 months); NB! less stable, than acidic phenol
NB! dangerous substance!

» add to accidic phenol 1x volume of 0.2 M Tris-base, mix ~0.5h;
» check pH of aqueous phase (pipette on the pH-strip), should be pH ~8;
» remove water phase;
» add and mix ~0.5h:
0.1M Tris-Cl, pH 8.00.1x V of phenol (~100ml per liter)
βMeEtOH (2-Mercaptoethanol)0.02% of phenol (~200µl per liter)
In some manuals authors recommend neutralize phenol by sequantial mixing with neutral Tris buffer. No advantages, and a lot of disadvantages:
» a lot of cycles, because phenol is a strong buffer
» slightly predictible (control pH of each equilibration)
» lose a lot (~1/3) of phenol (~7% of each aqueous phasephase is phenol)



[править] Chloroform/Isoamil alcohol

24:1
store at 4°C

conc.stock50ml
Chloroform24x100%48ml
Isoamil alcohol1x100%2ml



[править] Phenol/Chloroform/Isoamil alcohol

25:24:1
store at 4°C
NB! dangerous substance!

conc.stock50ml
Phenol1x100%25ml
Chloroform/Isoamil alcohol1x100%25ml




[править] Notes

  • useful information is on the Les Lane web site


Phenol

  • Phenol dissolves proteins and lipids leaving water soluble matter (carbohydrates, nucleic acids, etc.) in the aqueous phase. Phenol is less a denaturant than a solvent. The distribution coefficient of proteins into the phenol phase is ~100-1000.
  • To prevent losses of DNA during extraction:
» add salt to aqueous phase (at least 100mM)
» be sure, that pH>7: in the range 7.5-8.5
  • Phenol phase is a two-component solution: 28% water and 72% phenol. Aqueous phase: 7% phenol and 93% water.
  • Phenol stability: oxidizes by a free radical process. Free radical oxidation cleaves polynucleotide backbones. Oxidation acelerates with temperature and pH. Adding of 10mM HCl should reduce pH by 2-3 units increasing the lifetime of "clear phenol" by a factor of at least 100. Stability drops in order:
» cristalls, -20°C
» cristalls, room temperature
» H2O saturated, acidic, -20°C
» H2O saturated, acidic, 4°C
» H2O saturated, neutral, 4°C


Chloroform

  • Chloroform in Phenol/Chloroform mixture:
» antifreeze
» increases organic phase density (simplify aqueous phase transfer)
» improves phase separation (simplify aqueous phase transfer)
» seems to decrease solubility phenol in water, so somehow decreasing "extraction power" of pure phenol
  • extraction with Chloroform: removes phenol traces from aqueous phase during chloroform extraction
  • when it is necessary to perform phenol extraction first and when it is possible to start with phenol/chloroform extraction:
» "phenol extraction" should be more efficient, because more phenol will be in the aqueous phase. Normally used for primary extraction of NA from organisms (bacteria, tissue, etc.) and followed by second (more simple) phenol/chloroform extraction
» for purification of enzimatic reactions one phenol/chloroform extraction is enough


  • Isoamil alcohol is antifoam. It is the only function. In other respects it is optional for NA extraction.


  • 8-Hydroxyquinoline (chelator) and β-Mercaptoethanol are antioxidants (phenol stabilization). Besides, 8-Hydroxyquinoline:
» make phenol yellow (help to distinguish organic phase)
» weak RNase inhibitor


  • removal of phenol from aqueous phase:
» aqueous phase should not smell by phenol
» it is possible to remove the very traces of organic solvent by additional Ether extraction
» in critical cases it is possible to control UV spectrum of ethanol washes


Proteinase K digestion

  • it is better to use Proteinase K digestion, if you are not sure, that "everything fine without it". Obviously improve solubility of DNA pellet after some of enzymatic reactions: end-repair, EcoR1 restriction.
  • Digestion buffer for Proteinase K
» it is important, that pH>8
» SDS increase Prot.K activity and improves Phenol extraction
  • it is not necessary to perform "self digestion" of Proteinase K


Phase Lock

  • Phase Lock Tubes accelerate extraction and increase the output: ~95% w/o phase lock, ~98% with phase lock
  • if necessary, most of enzymatic reactions may be performed in the presence of phase lock gel
  • it is possible to prepare 0.5ml phase-lock tubes by dispensing (syringe) of PL-gel into tube caps (it is possible to buy phase lock gel dispensed in syringes)
  • MaXtract High Density tubes, Qiagen:
typefree volumemax. sample volumeCat#, priceamount of PL-gel
0.5ml0.45ml150µlself-dispensing 
1.5ml????200x, #129046, $157??
2ml1.7ml560µl200x, #129056, $16080-100ml
15ml????100x, #129065, $136??
50ml45ml15ml25x, #129073, $121~150ml




  1. there is enough salt in the ProtK buffer
  2. centrifugation of Phase Lock Tubes:
    » 1.5 and 2ml tubes: ~14ktpm
    » 15 and 50ml tubes: 4.5krpm
  3. 3,0 3,1 Phenol/Chloroform/Isoamil alcohol = 25:24:1; neutral
  4. 4,0 4,1 4,2 4,3 4,4 centrifugation of Phase Lock Tubes:
    » 1.5 and 2ml tubes: ~14ktpm, 5min.
    » 15 and 50ml tubes: 4.5krpm, 10min.
  5. 5,0 5,1 Chloroform/Isoamil alcohol = 24:1
  6. do not vortex
  7. for SDS-containing buffers (like ProtK buffer) use Ethanol, not Isopropanol
  8. Phenol; neutral
  9. 9,0 9,1 9,2 shaking of NA with organic solution:
    » NA > 6kb: shake by hand
    » NA < 6kb: vortex
  10. 10,0 10,1 try do not catch the interphase. For valuable samples it is worth to "reextract" interphase with a new portion of aqueous buffer:
    tubeextraction volume
    0.5ml50µl
    1.5ml150µl
    2ml150µl
    15ml1.5ml
    50ml3ml


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