Molecular Techniques and Methods

5'-end Labeling of DNA
with T4 Polynucleotide Kinase

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This procedure is useful for radioactive end labeling of oligonucleotides. The 5'-end of DNA should be dephosphorylated with alkaline phosphatase before kinase treatment. The alkaline phosphatase treatment described below can also be used to prevent recircularization and religation of linearized cloning vehicle DNA by removing phosphate groups from both 5'-termini.




MATERIALS AND SOLUTIONS

CIAP 10 x Buffer (1 ml)
500 mM Tris-HCl (pH9.0) ------------------------- 500 ul of 1 M Tris-HCl
10 mM MgCl2 ------------------------------------- 10 ul of 1 M MgCl2
1 mM ZnCl2 --------------------------------------- 1 ul of 1 M ZnCl2
100 mM Spermidine ------------------------------- 100 ul of 1 M Spermidine
Distilled H2O -------------------------------------- 389 ul


Kinase 1 x Buffer (1 ml)
500 mM Tris-HCl (pH 7.5) ----------------------- 500 ul of 1 M Tris-HCl
100 mM MgCl2 ----------------------------------- 100 ul of 1 M MgCl2
50 mM DTT -------------------------------------- 50 ul of 1 M DTT
1 mM Spermidine --------------------------------- 1 ul of 1 M Spermidine
Distilled H2O -------------------------------------- 349 ul




PROCEDURES

Dephosphorylation Reaction (from both 5'-termini of a linear molecule)

1. Prepare the following reaction in a microfuge tube.

CIAP 10 x Buffer
5 ul
Linear DNA (up to a total of 10 pmoles of 5'-ends)
1 ul
Calf Intestinal Alkaline Phosphatase (diluted in CIAP 1 x buffer)
0.5 ul
Add distilled H2O to make a final volume of
50 ul


For protruding 5'-termini dephospharylation:

2. Incubate at 37oC for 30 minutes.

3. Add another 0.1 units of alkaline phosphatase and incubate for another 30 minutes at 37oC.



For recessed 5'-termini/ or blunt end dephosphorylation:

2. Incubate at 37oC for 15 minutes, then at 560C for 15 minutes.

3. Add another 0.1 units of alkaline phosphatase and repeat incubations at both temperatures.



4. To stop the reaction, add 1 volume of TE-saturated phenol: chloroform: IAA.

5. Vortex for 1 minute and spin in a microcentrifuge at 13,000g for 5 minutes.

6. Remove the upper, aqueous phase to a fresh tube and repeat Step 4 and 5.

7. Transfer the upper, aqueous phase to a fresh tube.

8. Add 1 volume of Chloroform: Isoamyl alcohol (24:1).

9. Vortex for 1 minute and centrifuge in a microcentrifuge as in Step 5.

10. Transfer the upper, aqueous phase to a fresh tube.

11. Add 0.1 volume of 3M Sodium acetate (pH 7.0).

12. Add 2 volumes of 100% ethanol and 10 ug of glycogen as a career.

13. Centrifuge at 13,000g for 20 minutes.

14. Carefully pour off the supernatant.

15. Air-dry the pellet briefly.

16. Resuspend the DNA in 34 ul of Kinase 1 x Buffer.




Kinase Reaction

17. After the 5' phosphate groups have been removed, the substrate DNA can be labeled by kinase reaction.

18. To the reaction add the following:

[r-32P]ATP (3,000 Ci/ mmole at 10 mCi/ ml)
15 ul
T4 Polynucleotide kinase (at 8-10u/ ul)
1 ul
Add distilled H2O to make a final volume of
50 ul


19. Incubate at 37oC for 10 minutes.

20. Stop the reaction by adding 2 ul of 0.5M EDTA.

21. Add 1 volume of TE-saturated phenol: chloroform: IAA.

22. Vortex for 1 minute and centrifuge at 13,000g for 5 minutes.

23. Transfer the upper, aqueous phase to a fresh tube.

24. Add 0.5 volume of 7.5M Ammonium acetate and 10 ug of glycogen as a career.

25. Add 2 volumes of 100% ethanol.

26. Centrifuge at 13,000g for 20 minutes.

27. Redissolve the DNA in 50 ul of TE buffer.




NOTES

l pmole of 5'-ends of linear DNA (1,000 bp) is equivalent to
0.37 ug
l pmole of 5'-ends of linear DNA (2,000 bp) is equivalent to
0.74 ug
l pmole of 5'-ends of linear DNA (3,000 bp) is equivalent to
1.1 ug
l pmole of 5'-ends of linear DNA (4,000 bp) is equivalent to
1.48 ug
l pmole of 5'-ends of linear DNA (5,000 bp) is equivalent to
1.85 ug


  • Ammonium ions are strong inhibitors of bacteriophage T4 polynucleotidekinase; therefore, DNA should not be dissolved in, or precipitated from, buffers containing ammonium salts prior to treatment with kinase.


  • The concentration of ATP in the kinase reaction should be at least 1 uM.



  • KIT INFORMATION



    REFERENCES


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

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified