Molecular Techniques and Methods

Non-Radioactive Labeling of DNA by Nick Translation

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This procedure incorporates one modified nucleotide (DIG-dUTP, etc..) at every 20-25th position in the newly synthesized DNA. This labeling density allows optimal enzymatic incorporation of the modified nucleotide and produces the most sensitive probes for indirect, immunological detection.




MATERIALS AND SOLUTIONS

10 x Nick Translation Buffer (1 ml)
500 mM Tris-HCl (pH 7.8) ------------------------- 500 ul of 1 M Tris-HCl
50 mM MgCl2 -------------------------------------- 50 ul of 1 M MgCl2
Bovine Serum Albumin (nuclease-free) -------------- 0.5 mg
Add deionized H2O to make a final volume of ----- 1 ml


Nucleotide Mixture (1 ml)
0.5 mM dATP ------------------------------------- 5 ul of 100 mM dATP
0.5 mM dGTP ------------------------------------- 5 ul of 100 mM dGTP
0.5 mM dCTP ------------------------------------- 5 ul of 100 mM dCTP
0.1 mM dTTP ------------------------------------- 1 ul of 100 mM dTTP
Deionized H2O ------------------------------------ 984 ul




PROCEDURES

1. Add the following components to the microcentrifuge tube on ice.

10 x Nick Translation Buffer ------------------------ 5 ul
100 mM Dithiothreitol ------------------------------- 5 ul
Nucleotide Mixture ---------------------------------- 4 ul
1 mM DIG-dUTP ----------------------------------- 2 ul
Template DNA -------------------------------------- 1 ug
DNase I (1 ng/ul) ------------------------------------ 5 ul
DNA Polymerase I (10 U/ul) ------------------------ 1 ul
Add sterile H20 to make a
final volume of ---------- 50 ul

2. Incubate at 16oC for 2 hour.

3. Chill the reaction tube on ice.

4. Take a 3 ul aliquot from the tube and run the sample on an agarose minigel with a DNA molecular weight marker.

5. Depending on the average size of the probe, do one of the following:

  • If the probe is between 200 and 400 nucleotides long, go to Step 6.


  • If the probe is longer than 400 nucleotides, incubate the reaction tube further at 16oC until the fragments are the proper size.


  • 6. To the tube, add carrier DNA (e.g., 50-fold excess of fragmented herring sperm DNA) and carrier RNA (e.g., 50-fold excess of yeast tRNA).

    7. Add 0.1 volume 3 M Sodium acetate (pH 5.6) and 2.5 volumes prechilled (-20oC) 100% ethanol.

    8. Mix reagents by inverting tubes. Do not use pipette tip to mix.

    9. Place tube on ice for 30 minutes.

    10. Centrifuge tube 13,000g for 30 minutes at 4oC.

    11. Discard the supernatant.

    12. Dry the pellet under vacuum.

    13. Dissolve the pellet in a minimal amount of TE (pH 8.0) buffer, then dilute an aliquot of the probe solution to a convenient stock concentration (e.g., 10-40 ng/ul) in the hybridization buffer to be used for the in situ hybridization.

    (If you are not going to use the probe immediately, dissolve the pellet in a minimal amount of TE (pH 8.0) buffer and store the probe solution at -70oC.)



    NOTES

  • DNA does not need to be denatured before it is labeled by nick translation.


  • For in situ hybridization procedures, the length of the labeled fragments obtained from this procedure should be about 200-500 bases.


  • Instead of DIG-dUTP, 2 ul of 1 mM Biotin-dUTP or, 1 mM fluorochrome-labeled dUTP can be used in the nick translation reaction.


  • If the fragment is too long, the labeled probe can also be sonicated to the proper size.


  • 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.


  • Avoid repeated freezing and thawing of the probe.





  • KIT INFORMATION




    REFERENCES

  • Langer, P.P., Waldrop, A.A., and Ward, D.C., 1981, Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. PNAS, 78, 6633-6637.


  • Rigby, P.W., Dieckman, M., Rhodes, C., and Berg, P., 1977, Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-241.


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

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