Molecular Techniques and Methods

3'-end Labeling of Oligonucleotides
with Terminal Transferase

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Terminal deoxynucleotidyl transferase catalyzes the repetitive addition of mononucleotides from a dNTP to the terminal 3'-OH of a DNA initiator, accompanied by the release of inorganic phosphate. The enzyme provides a unique method for the labeling of the 3'-termini of DNA with 32P for subsequent utilization in hybridization assays.




MATERIALS AND SOLUTIONS

Terminal Transferase 5 x Buffer
500 mM Cacodylate (pH6.8)
1 mM CoCl2
0.5 mM DTT
500 ug/ ml BSA




PROCEDURES

1. Set up the following reaction:

5 x Terminal Transferase Buffer
4 ul
Oligonucleotides to be labelled (see NOTES)
2 pmole
[a-32P] dCTP (800 Ci/mmole, l0 mCi/ ml)
1.6 ul
Terminal transferase (10-20u/ ul)
1.0 ul
Add deionized H2O to make a final volume of
20 ul


2. Incubate at 37oC for 60 minutes.

3. Stop the reaction by heating at 70oC for 10 minutes.

4. Remove unincorporated [a-32P]dCTP.




NOTES


15-mer oligonucleotides
10 ng/ 2 pmole
18-mer oligonucleotides
12 ng/ 2 pmole
24-mer oligonucleotides
16 ng/ 2 pmole
31-mer oligonucleotides
21 ng/ 2 pmole


  • Incorporation can also be limited to a single nucleotide by using [a-32P]cordycepin-5'-triphosphate (instead of [a-32P]dCTP). This analog lacks a free 3'-hydroxyl group, preventing incorporation of additional nucleotides.



    • KIT INFORMATION



    REFERENCES

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