DNA purification from tissue

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NB! pre-PCR operation


[править] Protocol for large amount of tissue (>0.1g)

    • check, that you have:
    » liquid nitrogen
    » mortar and pestle
    » (desirable) phase-lock gel
    • it is necessary to know the weight of the tissue

    [править] Homogenization

  1. select mortar:
    weight of the tissue [g]mortar diameter [cm]
    >1.516
    0.5-1.512.5
    <0.511

  2. NB! tissue disruption should be done unde fume hood[1]

  3. put mortar on foam plastic lid;
  4. precool mortar and pestle with liquid nitrogen;
  5. freeze tissue in liquid nitrogen directly in mortar;
  6. grind in tiny powder;</li>
  7. add liquid nitrogen[2] and desired volume of DB: normally, ~12ml per 1g of tissue (see also table in Notes);
  8. grind in tiny powder;
  9. transfer powder into a tube;

  10. melt[3];
  11. thoroughly mix [4];
  12. incubate at 56°C for more than 3h[5];

  13. [править] Organic solvent extraction

    NB! see "Notes" about mixing and transfer of aqueous phase
    use LIGHT phase-lock gel for phenol extraction
  14. neutral phenol extraction:
    • add 1x V of Ph[6] and mix
    • centrifuge 1.5krpm, room temperature, 3min.[7]
    • centrifuge room temperature:
    » up to 2ml tube: 14krpm, 5min.
    » 15, 50ml tubes: 4krpm, 10min.
    • transfer the upper (aqueous) phase to a new tube

  15. phenol/chlorohorm extraction:
    LIGHT or HEAVY phase-lock gel
    • add 1x V of Ph/Ch/I[8] and mix
    • centrifuge room temperature:
    » up to 2ml tube: 14krpm, 5min.
    » 15, 50ml tubes: 4krpm, 10min.
    • transfer the upper (aqueous) phase to a new tube

  16. chloroform extraction:
    LIGHT or HEAVY phase-lock gel
    • add 1x V of Ch/I[9], vortex for 1-3min.
    • centrifuge room temperature:
    » up to 2ml tube: 14krpm, 5min.
    » 15, 50ml tubes: 4krpm, 10min.
    • transfer the upper (aqueous) phase to a new tube

  17. [править] Ethanol precipitation

    see "Notes" about selection of purification method

    see also: Ethanol precipitation

    procedure for ~60kb

  18. add 2.5x V of Ethanol 96% (or 1x V of Isopropanol), mix;
  19. incubate 5-10min. at room temperature;
  20. centrifuge:
    » up to 2ml tube: room temperature, 14krpm, 5min.
    » 15, 50ml tubes: 4°C, 4krpm, 10min.
  21. wash pellet with 70% EtOH;
  22. solubilize in EB: 2-10µl per mg of tissue;
  23. determine DNA concentration on Qubit, BR kit;
  24. snap freeze in liquid nitrogen, store at -20°C




[править] Quick protocol for small pieces of tissue (<200mg)

for biopsies, <1cm tails, pierces of skeen, mouth swabs, etc.

  1. digestion:
    • put sample into 2ml tube with 500µl of DB with Protinase K
    • incubate 3n-overnight in Eppendorf thermoshaker: 56°C, 300rpm[10]
  2. organic solvent extraction:
    • 0.5ml Ph[6] extraction in 2ml tube with LIGHT phase-lock gel
    • 0.5ml Ph/Ch/I[8] extraction in 2ml tube with HEAVY phase-lock gel
    • 0.5ml Ch/I[9] extraction in the same 2ml tube
  3. Ethanol precipitation:
    • transfer supernatant into 2ml tube with 1.25ml of 96% Ethanol and mix
    • incubate 5-10min. at room temperature
    • centrifuge 14krpm, room temperature, 5min.
    • wash pellet once with 300µl of 70% EtOH
  4. storage:
    • solubilize in EB: 2-10µl per mg of tissue
    • determine DNA concentration on Qubit, BR kit
    • snap freeze in liquid nitrogen, store at -20°C




[править] Solutions

[править] DB (Digestion Buffer)

store at 4°C

conc.stock50ml
NaCl0.1M5M1.0ml
Tris Cl, pH 8.2-8.310mM1M0.5ml
EDTA25mM0.5M2.5ml
SDS0.5%10%2.5ml
H2O mQ43.5ml
Proteinase K*0.1mg/mlsolid
20mg/ml
5mg
250µl

 * add Proteinase K just before use

[править] DB (Digestion Buffer)

link too the protocol of preparation: Neutral phenol


[править] Notes

  • see also
» Phenol/Chloroform_purification
» Ethanol_precipitation



organismtissueDNA content
Drosophilawhole fly, mail0.75µg/fly
whole fly, femail1µg/fly
Mouseliver, kidney or lungs3-5µg/mg
skeletal muscle, heart or brain1-3µg/mg
tail, ~1cm10-30µg
Rattail, ~0.5cm20-40µg
Plant 0.3-0.8µg/mg



  • genomic DNA is extremely long. It is impossible to prepare it in intact form using this protocol . Shearing is strongly depends on "transfer of aqueous phase", "mixing with organic solvents", "final purification" and "storage". The longer extracted DNA should be, the more difficult extraction procedure. on practice, very long DNA is required only for long-insertion phage- or BAC-libraries.
fragment lengthtransfer of aqueous phasemixing with organic solventsfinal purificationstorage
>1000kbDNA purification in agarose blocks+4°C[11]
~200kbcutted tipslow (~10rpm) rotation for ~20min.dialysis: 1-2days against 5-7 changes of ~100x volume of TE
~150kb(~60rpm) rotation for ~10min.transfer clog into 70% EtOH w/o centrifugation-20°C
~60kbnormal tipintense mixing by handEthanol precipitation
~20kbvortexing




  1. it is possible to catch a strong alergy if do it on the table
  2. be accurate, powder is volatile
  3. at 20-56°C
  4. solition is viscous, mixing takes 3-5min.
  5. it is possible to leave overnight at 37°C
  6. 6,0 6,1 neutral phenol
  7. low-speed centrifugation to pellet solid particles (hair, bones, etc.) through the viscous DNA solution
  8. 8,0 8,1 Phenol/Chloroform/Isoamil alcohol = 25:24:1; neutral
  9. 9,0 9,1 Chloroform/Isoamil alcohol = 24:1; neutral
  10. put folded paper towel between tube caps and thermoshaker lid for thermoisolation
  11. bacterial/fungal growth is possible at +4°C. Plan the experiment to minimize storage at this temperature


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