Molecular Techniques and Methods

Non-Radioactive Labeling of
3'-end Oligonucleotides by Terminal Transferase

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

A single modified dideoxyuridine-triphosphate (e.g., DIG-ddUTP) is added to the 3'-ends of an oligonucleotide (HPLC- or gel-purified; 15-100 nucleotides long) by terminal transferase.




MATERIALS AND SOLUTIONS

Purified Oligonucleotide in Sterile H20


1 mM Solution of DIG-ddUTP in Sterile H20
(or Biotin-ddUTP, or Fluorescein-ddUTP)


5 x Reaction Buffer (1 ml)
1 M Cacodylate-Sodium salt ----------------------- 160 mg
0.125 M Tris-HCl --------------------------------- 125 ul of 1 M Tris-HCl
Bovine Serum Albumin ---------------------------- 1.25 mg
Distilled H2O to make a final volume of ---------- 1 ml
  • Adjust pH to 6.6.


    Reaction Stop Buffer (1 ml)
    0.2 M EDTA (pH 8.0) ------------------------------ 400 ul of 0.5 M EDTA
    Glycogen solution (10 ug/ ul) ------------------------ 10 ul
    Distilled H2O ---------------------------------------- 590 ul




    PROCEDURES

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

    5 x Reaction Buffer ---------------------------------- 4 ul
    25 mM CoCl2 ---------------------------------------- 4 ul
    Oligonucleotide to be labeled ------------------------- 100 pmole
    1 mM DIG-ddUTP ----------------------------------- 1 ul
    Terminal Transferase (50 U/ul) ------------------------ 1 ul
    Add distilled H20 to make a final volume of ------------ 20 ul


    2. Incubate the tube at 37oC for 15 min.

    3. Place the tube on ice.

    4. Add 2 ul of Reaction Stop Buffer to stop the reaction.

    5. Add 2.5 ul of 4 M LiCl and 75 ul of prechilled 100% ethanol.

    6. Let the precipitate form for at least 30 min at -70oC or 2 h at -20oC.

    7. Centrifuge the tube at 13,000g for 15 minutes (at 4oC).

    8. Discard the supernatant.

    9. Wash the pellet with ice-cold 70% (v/v) ethanol.

    10. Centrifuge the tube at 13,000g for 15 minutes (at 4oC).

    11. Discard the supernatant.

    12. Briefly air dry the pellet.

    13. Dissolve the pellet in a minimal amount of sterile, redistilled H2O

    14. Store the probe solution at -20oC. Caution: Avoid repeated freezing and thawing of the probe.

    15. Check the labeling efficiency by spotting the serial dilution of labeling reaction and a standard on the nylon membranes.




    NOTES

  • Do not increase the concentration of oligonticleotide in the labeling reaction. To make larger amounts of labeled oligonucleotide, scale up all reaction components and volumes.


  • Instead of DIG-ddUTP, same amount of Biotin-ddUTP, or Fluorescein-ddUTP can be added in the labeling reaction.


  • Do not use phenol: CHCl3: IAA extraction to stop the reaction, since the labeled oligonucleotide will migrate to the organic layer during such extraction.


  • The labeled oligonucleotide can be analyzed by polyacrylamide-gel electrophoresis and subsequent silver staining in comparison to the unlabeled oligonucteotide. DIG-labeled oligonucleodes are shifted to higher molecular
    weight due to the addition of the label.





  • KIT INFORMATION




    REFERENCES

  • Schmitz, G.G., Walter, T., Seibl, R., and Kessler, C., 1991, Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222-231.



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