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Protocol 2
Purification of mRNA


1. Preparations
  1. 4 M Guanidine thiocyanate
Guanidine thiocyanate (M.W. 118.16) 472.6 g
DEPC-treated water 500 ml

Add DEPC-water to 1000 ml @

  • 10% SDS
    SDS 10 g
    DEPC-water 60 ml

    Add DEPC-water to 100 ml

  • Homogenize buffer
    0.2 M Tris-HCl@Tris HCl salt (M.W. 157.6) 3.14 g
    0.2 M NaCl; NaCl (M.W. 59) 1.16 g
    2% SDS; @10% SDS 20 ml

    Add 60 ml of DEPC-water
    Adjust pH to 8.2 with 2N NaOH at 22Ž.
    Add DEPC-water to 100 ml (Leave at R.T.)

  • Wash buffer 500 ml
    @@ 0.05 M Tris-HCl;@Tris HCl salt (M.W. 157.6) 3.925 g
    @@ 0.5 M NaCl;@@NaCl (M.W. 59 g) 14.5 g
    @@ 0.1% SDS;@@10% SDS 5 ml

    @@ Add 450 ml of DEPC-water
    Adjust pH 7.5 with 2N NaOH at 22Ž
    Add DEPC-water to 500 ml

  • 5 M NaCl 100 ml
    @@ 5 M NaCl;@@NaCl (M.W. 59 g) 29.5 g
    @@ Add 80 ml of DEPC-water @

    @@ Add DEPC-water to 100 ml

  • Proteinase K buffer solution
    10 mM Tris-HCl, pH 8.0, 1 mM CaCl2
    @@ 1 M Tris stock solution, pH 8.0 1 ml
    @@ CaCl2@ (M.W. 110.98) 11 mg

    Add 100 ml of DEPC water and autoclave

    2. Purification
    1. Set a incubator at 45Ž.
    2. Proteinase K stock solution
    Proteinase K (Wako; 166-14003, 1 g, 82,000 yen) 10 mg X (sample #)
    Proteinase K stock buffer solution 1 ml X (sample #)

    preincubate at 37Ž for 15 min more before use (autodigestion of RNase).
    1. Prepare a pump for perfusion, Falcon tubes, homogenizer, masure etc.
    2. Wash the tube of pump with DEPC-water
    3. Warm the homogenize buffer (before adding proteinase K) at 37Ž, or 45Ž.
    4. Perfuse a rat via heart with 10 mM DEPC-treated PBS and then with 4 M guanidin thicyanate at 4Ž.
    5. Dissect tissues (about 500 mg) and cut them carefully.
    6. Mix 1 ml proteinase K stock solution containing10 mg of proteinase K and 9 ml of the homogenize buffer.
    7. Homogenize the tissues in 10 ml the proteinase K-homogenize buffer using polytron PT10 at setting 3 for 20 sec - 2 min or the tissue completely disrupted.
    8. Incubate the homogenates in shaking bath at 45Ž for 60 min -120 min.
    9. Adjust NaCl concentration from 0.2 M to 0.5 M with 5 M NaCl.
      10. Add 0.6 ml of 5 M NaCl to10 ml solution or
      Add 0.7 ml of 5 M NaCl to12 ml solution, and mix thoroughly.
    10. Add 20 - 30 mg of Oligo(dT)-cellulose stock (a capacity sufficient to bind 100 mg RNA). Mix gently on rocking platform for 30 - 60 min at R.T..
    11. Spin at 3,500 rpm (2,000 g) for 15 min at R.T..
    12. Wash the pellet two times with 10 ml of the wash buffer.
    13. Resuspend the pellet with 500 ul of the wash buffer.
    14. Transfer into a spin column.
    15. Spin 15,000 rpm (9,000 g) for 10 seconds at 4Ž.
    16. Wash the pellet 2-3 times until A260 < 0.05 with 300 ul of washing solution.
    17. Elute two times with 250 ul DEPC-water.
    18. Check the eluate at A240-320 (five times dilution).

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    Copyright (C) Central Research Laboratory. All right reserved.since 1996/2/1

    Last Updated 2005/8/8