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dsDNA can be in vitro radiolabeled with 32P by treatment with DNase I, DNA polymerase I, dNTPs, and a32P-dCTP. Several commercial kits are available for nick translation, but some laboratories prefer to nick translate from scratch to improve incorporation and save on costs. Of course, strict precautions must be used, as whenever mCi quantities of 32P is used.

  1. Materials

    1. DNase I storage buffer
      Final Conc.
      1ml 1M Tris, pH 7.5 50mM
      200ul 0.1M DTT 1mM
      200ul 1M MgSO4 10mM
      10ml glyerol 50%
      20ml filter sterilize

    2. DNase I dilution buffer
      Final Conc.
      1ml 1M Tris, pH 7.5 50mM
      200ul 1M MgSO4 10mM
      200mM 0.1M DTT 1mM
      20ml filter sterilize

    3. 10X NT buffer
      Final Conc.
      5ml 1M Tris,pH 7.5 0.5M
      1ml 1M MgSO4 0.1M
      1ml 0.1M DTT 10mM
      5mg BSA (nuclease-free) 0.5mg/ml
      10ml

    4. dNTP mix
      0.2mM @ dTTP, dCTP & dGTP, each from 2mM stocks. For labeling with alpha-32P dNTPs besides dATP, use the other 3 dNTPs in the dNTP mix.

    5. Stop sol'n
      Final Conc.
      4ml 0.1M Na3 EDTA 20mM
      4ml 10mg/ml sssDNA 2 mg/ml
      20ml see hybridization Rx for sssDNA recipe

    6. TE
      Final Conc.
      10ml 1M Tris, pH 7.5 10mM
      10ml 0.1M EDTA 1mM
      liter

    7. Column dye
      Final Conc.
      50 mg bromophenol blue 0.25%
      10ml glycerol 50%
      10ml 1X TE 1/2 X
      20ml

  2. Preparation of DNase I

    1. Add 0.5ul 1mg/ml stock DNase I to 100ul dilution buffer on ice.
    2. Add 0.5ul diluted DNase I to 100ul dilution buffer, again on ice. Final dilution is 1:40,000.

  3. Nick translation

    1. Mix the following ingredients on ice:
      2.5ul 10X NT buffer
      2.5ul dNTP mix
      50uCi alpha-32P-dATP
      0.5ul diluted DNase I
      X ul DNA (1ug)
      Y ul ddH2O (to 20ul)

    2. Add 0.1ul 2mg/ml DNA polymerase I. Mix and incubate at 14C for 3 hrs.
    3. Add 25ul Stop sol'n.

  4. Column chromatography

    1. Add 50ul TE and 10ul column dye to the sample.
    2. Apply the sample onto a 0.7 X 20 cm G50 sephadex column in TE.
    3. Run the column with TE, collecting the first 20 fractions, 12 drops each. Wash the column in TE until the blue dye is all washed out of the column.
    4. Spot 2ul of each fraction onto 4-5mm squares of 3MM paper. Count each square in 4ml scintillation fluid.
    5. Pool the G50 excluded peak (i.e. the first peak), which usually is at fractions 7-10. Store at -20C until use.


CSH 'Molecular cloning' 1983
CSH molecular cloning workshop, attended by P. Hamilton
Paul Hamilton, personal communication

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