Molecular Techniques and Methods

Nuclear Run-Off Analysis
Adherent Mammalian Cells

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Lysis Buffer (50 ml)
10 mM Tris-HCl (pH 7.4) ----------------------- 500 ul of 1 M Tris-HCl
10 mM NaCl --------------------------------------- 100 ul of 5 M NaCl
3 mM MgCl2 -------------------------------------- 150 ul of 1 M MgCl2
DEPC-treated H2O ------------------------------ 48.75 ml
  • Autoclave and store at 4oC.

    0.5% NP-40 (Igepal) ---------------------------- 500 ul of 50% NP-40 (Igepal)
  • Add NP-40 just prior to use.



    Nuclear Storage Buffer (10 ml)
    50 mM Tris-HCl (pH 8.3) ----------------------- 500 ul of 1 M Tris-HCl
    5 mM MgCl2 --------------------------------------- 50 ul of 1 M MgCl2
    40% (V/v) Glycerol ------------------------------ 8 ml of 50% Glycerol
    0.1 mM EDTA ----------------------------------- 2 ul of 0.5 M EDTA
    DEPC-treated H2O ------------------------------ 1.448 ml
  • Autoclave and store at 4oC.


    5x Transcription Buffer (1 ml)
    20 mM Tris-HCl (pH 8.0) ----------------------- 20 ul of 1 M Tris-HCl
    20 mM MgCl2 ------------------------------------- 20 ul of 1 M MgCl2
    0.6 M KCl ------------------------------------------ 600 ul of 1 M KCl
    10 mM DTT --------------------------------------- 10 ul of 1 M DTT
    2 mM ATP ----------------------------------------- 20 ul of 100 mM ATP
    2 mM CTP ----------------------------------------- 20 ul of 100 mM CTP
    2 mM GTP ----------------------------------------- 20 ul of 100 mM GTP
    0.04 mM UTP -------------------------------------- 4 ul of 10 mM UTP
    DEPC-treated H2O ------------------------------ 8.38 ml
  • Store at -20oC.


    HSB Buffer (10 ml)
    0.5 M NaCl --------------------------------------- 1 ml of 5 M NaCl
    50 mM MgCl2 ------------------------------------- 500 ul of 1 M MgCl2
    2 mM CaCl2 ------------------------------------- 20 ul of 1 M CaCl2
    10 mM Tris-HCl (pH 8.0) --------------------- 100 ul of 1 M Tris-HCl
    DEPC-treated H2O ------------------------------ 176 ul


    SDS-Tris Buffer (12.5 ml)
    5% (w/v) SDS -------------------------------- 5 ml of 10% SDS
    0.125 M EDTA -------------------------------- 2.5 ml of 0.5 M EDTA
    0.5 M Tris-HCl (pH 7.4) --------------------- 5 ml of 1 M Tris-HCl


    Hybridization Buffer (10 ml)
    5 mM TES (pH 7.4) ---------------------------- 500 ul of 1 M TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]
    5 mM EDTA ------------------------------------ 100 ul of 0.5 M EDTA
    0.1% SDS -------------------------------------- 100 ul of 10% SDS
    DEPC-treated H2O ------------------------------ 9.3 ml





    PROCEDURES

    Preparation of Nuclei

  • Through out the procedure, cells should be kept in ice to prevent protein degradation.

    1. Grow up cells to confluence in 5-7, 10 cm plates.
  • At least 5 x 107 cells/assay needed.

    2. Change medium 24 hr prior to harvest.

    3. Wash cells with cold PBS. 2x.

    4. Harvest cells by scrapping and centrifugation at 500g for 5 min.

    5. Wash cells with cold PBS.

    6. Resuspend in 4 ml cold Lysis Buffer (NP-40 added just prior to use) by gentle vortexing.

    7. Let sit on ice for 5 min.

    8. Pellet nuclei at 500g for 5 minutes.

    9. Remove the supernatant.

    10. Wash nuclei in 4 ml Lysis Buffer by gentle vortexing and centrifugation at 500g for 5 min. 2x

    11. Resuspend the nuclei in 200 ul of Nuclear Storage Buffer and mix by pipetting up and down several times with a yellow tip.

    12. Freeze immediately at -70oC even if you continue with the next steps.
  • Upon thawing, the quality of the nuclei becomes obvious.
  • The nuclei should be dispersed easily by pipetting with a yellow tip. Big clumps, which are difficult to disperse, indicate bad quality.
  • Nuclei is stable for >1 year.


    Nuclear Transcription

    13. Add the followings in a 15 ml tube.

    5x Transcription Buffer
    50 ul
    [32P]-UTP (800 Ci/mmole of 10 mCi/ml: NEN #BLU007H)
    10 ul
    1 M DTT
    1 ul
    RNase inhibitor (40 U/ul)
    3 ul
    Nuclei
    200 ul

  • Vortex briefly and gently.

    14. Incubate for 30 minutes at 30oC. Shake every 10 minutes.

    15. Add 24 ul RNase-free DNase I (1 ug/ul) and 600 ul of HSB Buffer.
  • Pipete up and down 15 times to mix thoroughly.

    16. Incubate for 5 min at 30oC.

    17. Add 200 ul SDS-Tris Buffer and 10 ul Proteinase K (20 ug/ul).

    18. Incubate for 30 min at 42oC.


    Preparation of Labeled RNA

    19. Add 50 ul of 3 M NaOAc (pH 4.8), 2 ul of glycogen (10 ug/ul) and 500 ul phenol:CHCl3.

    20. Extract RNA. 2x

    21. Extract RNA with CHCl3:IAA.

    22. Add the 600 ul of iPr-OH.

    23. Centrifuge at 10,000g, 4oC for 30 minutes.

    24. Rinse the RNA with 70% ethanol.

    25. Resuspend the pellet in 100 ul DEPC-treated H2O.

    26. RNA should be denatured at 90oC for 5 min just prior to adding to hybridization solution.



    Filter Hybridization

    27. Amplify the inserts out of the cloning vector using primers specific for flanking vector sequences.

    28. Denature 2 ug of each fragment (per filter) with 0.25 N NaOH for 10 minutes at room temperature and apply via individual slots to a nylon membrane in a slot blot device.

    29. Immediately wash the slots with 1 M Ammonium acetate (5-10 volumes) to neutralize the DNA solution.
  • Prepare identical strips with multiple samples for each hot RNA probe.

    30. UV cross-link DNA on nylon membrane.

    31. In 5 ml scintillation vial, prehybridize membrane in 2 ml ULTRAhyb (Ambion AM8670) for 1 hr at 42oC.
  • ULTRAhyb should be pre-warmed for 30 min to prevent precipitation.

    32. Add radiolabeled RNA (1 x 106 cpm/mL) and hybridize at 42-55oC for 24 hr.
  • Use the same amount of label for each nuclear RNA preparation.
  • If using TES Hybridization Buffer, incubate for 36 hr at 65oC.
  • Radiolabeled RNA should be freshly prepared.

    33. Wash membrane with 2x SSC and 0.1% SDS at 42oC for 20 min. 3x.

    34. If high background observed, wash membrane again with 5x SSC and 0.5% SDS at 68oC for 15 min. 2x.

    35. Expose the membrane to phosphoimmager.


    NOTES


    KIT INFORMATION


    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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