Molecular Techniques and Methods

Non-Radioactive Labeling of DNA by Random Priming

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This method allows efficient labeling of 10 ng-3 ug DNA. This labeling method works with both short DNA (200 bp) and long DNA (cosmid or lamda DNA). The standard labeling reaction is fast (1 hour) and incorporates one modified nucleotide (DIG-dUTP, biotin-dUTP, or fluorescein-dUTP) at every 20-25th position in the newly synthesized DNA probe. This labeling density produces the most sensitive probes for indirect, immunological detection.




MATERIALS AND SOLUTIONS

5 x Labeling Buffer (1 ml)
500 mM Tris-HCl (pH 7.2) ---------------------- 500 ul of 1 M Tris-HCl
100 mM MgSO4 --------------------------------- 100 ul of 1 M MgSO4
1mM DTT --------------------------------------- 1 ul of 1 M DTT
Random hexadeoxyribonucleotides --------------- 26 A260 units
Add distilled H2O to make a final volume of ---- 1 ml
  • Store at -20oC.


    5 x dNTP Mix (1 ml)
    1 mM dATP ------------------------------------- 10 ul of 100 mM dATP
    1 mM dCTP ------------------------------------- 10 ul of 100 mM dCTP
    1 mM dGTP ------------------------------------- 10 ul of 100 mM dGTP
    0.65 mM dTTP ---------------------------------- 6.5 ul of 100 mM dTTP
    0.35 mM DIG-dUTP ---------------------------- 350 ul of 1 mM DIG-dUTP
    Sterile H2O -------------------------------------- 613.5 ul




    PROCEDURES

    1. To a 1.5 ml microcentrifuge tube, add the following components.

    Template DNA ---------------------------- 1 ug
    Sterile H20 --------------------------------- 6 ul

    2. Denature the DNA in a boiling water bath for 5-10 minutes.

    3. Chill the tube quickly in ice.

    4. Add the following components.

    5 x Labeling Buffer ----------------------- 4 ul
    50% Glycerol ----------------------------- 4 ul
    5 x dNTP Mix --------------------------- 4 ul
    Klenow Enzyme (1 U/ul) ------------------ 2 ul


    5. Incubate the tube at least 1 hour at 37oC.

    6. To stop the reaction, add 2 ul 0.2 M EDTA (pH 8.0) to the reaction tube and/or heat the tube to 65oC for 10 min.

    7. To the labeled DNA, add 2.5 ul 4 M LiCl and 75 ul prechilled (-20oC) 100 % ethanol.

    8. Let the precipitate form for at least 30 minutes at -70oC or 2 hour at -20oC.

    9. Centrifuge the tube (at 13,000g) for 15 min at 4oC.

    10. Discard the supernatant.

    11. Wash the pellet with 50 ul ice-cold 70% (v/v) ethanol.

    12. Centrifuge the tube (at 13,000g) for 5 min at 4oC.

    13. Discard the supernatant.

    14. Dry the pellet under vacuum.

    15. Dissolve the pellet in a minimal amount of TE (pH 8.0) buffer.

    16. Dilute an aliquot of the probe solution to a convenient stock concentration (e.g., 10-40 ng/ml) in the hybridization buffer to be used for the in situ experiment.

  • (If you are not going to use the probe immediately, store the probe solution at -20oC.)





  • NOTES

  • The amount of newly synthesized labeled DNA depends on the amount and purity of the template DNA, the label used, and the incubation time. Longer incubations (up to 20 hours) increase the yield of labeled DNA.


  • The size of the labeled DNA fragments obtained by random primed DNA labeling depends on the amount and size of the template DNA. With linear pBR322 plasmid DNA, the standard (1 hour) reaction produces labeled DNA fragments which are approximately 200-1000 bp long.


  • Linear DNA is labeled more efficiently than circular and supercoiled DNA. In all cases the template DNA should be thoroughly heat-denatured prior to random primed labeling. DNA fragments in low melting point agarose are also labeled efficiently, but often with slower kinetics.


  • For this standard reaction, the amount of DNA in the 6 ul sample can vary from 10 ng to 3 ug of DNA. To label > 3 ug of DNA, scale up the volume of the sample as well as the amounts and volumes of all reaction components.

  • Caution: Complete denaturation is essential for efficient labeling.

  • Instead of DIG-dUTP in 5 x dNTP Mix, biotin-dUTP or fluorescein-dUTP can be used for labeling.


  • Drying the pellet is important because small traces of residual ethanol will cause precipitation if the hybridization mixture contains dextran sulfate. Trace ethanol can also lead to serious background problems.


  • The DIG-dUTP is alkali-labile. Avoid exposing DIG-labeled probes to strong alkali (e.g., 0.2 mM NaOH).



  • KIT INFORMATION




    REFERENCES

  • Feinberg, P., and Vogelstein, B.,1984, A technique for radiolabeling DNA restriction enzyme fragments to high specific activity. Anal. Biochem. 137, 266-267.


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

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