Description

T4 polynucleotide kinase catalyzes the transfer and exchange of phosphate groups from the γ-position of ATP to the 5’–hydroxyl terminus of nucleic acids (double and single-stranded DNA and RNA). It can be used for the phosphorylation of DNA fragments or PCR primers. The efficiency of the reaction is affected by the status of the ends of the DNA strand (i.e., 3’-overhang, 5’-overhang, blunt or single strand).

Features

  • Two different reaction buffers for different 5’-ends of nucleic acids are included in the kit.
  • Highly purified enzyme from a recombinant source

Applications

  • Phosphorylates the 5’-ends of DNA/RNA fragments (e.g., PCR product or primer)
  • End-labels the 5’-end of DNA fragments using [γ-32P] rATP

Source

E. coli strain carrying the cloned T4 polynucleotide kinase gene

Unit definition

One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmole of γ-phosphate from [γ-32P]rATP in an acid-insoluble material in 30 min at 37ºC.

Storage conditionn

50 mM Tris-HCl (pH7.5), 0.1mM EDTA, 1mM DTT, 50 mM KCl, 0.1 mM rATP, 50% Glycerol
Store at -20ºC

Components

T4 Polynucleotide Kinase (2-20U/µl)
10 X Protruding End Kinase Buffer
10 X Blunt End Kinase Buffer
Denaturation Buffer

Principle

Typical reaction set up

<Phosphorylation of double strand DNA having 5’ overhanged ends>

Component Amount
DNA -1 µg
10 x Protruding End Kinase Buffer 5 µl
10 mM rATP* 5 µl
T4 Polynucleotide Kinase (5-20 U/µl) X µl
Distilled water Y µl
Total reaction volume 50 µl

*Caution: do not use dATPs. This reagent is not supplied with this product.

<Reaction condition>

37ºC, 60 min.

<Phosphorylation of double strand DNA having 3’ overhanged and blunt ends>

Component Amount
DNA -1 µg
10 x Blunt End Kinase Buffer 5 µl
10 mM rATP* 5 µl
T4 Polynucleotide Kinase (5-20 U/µl) X µl
Distilled water Y µl
Total reaction volume 50 µl

*Caution: do not use dATPs. This reagent is not supplied with this product.

<Reaction condition>

37ºC, 60 min.

<Phosphorylation of Oligo nucleatide>

Component Amount
Oligo nucleotide (50 µM) 2 µl
10 x Protruding End Kinase Buffer 2 µl
10 mM rATP* 2 µl
T4 Polynucleotide Kinase (5-20 U/ml) X µl
Distilled water Y µl
Total reaction volume 20 µl

*Caution! do not use dATP. This reagent is not supplied with this product.

<Reaction condition>

37ºC, 60 min.
95ºC, 5 min.

Add 50 µl distilled water

Use this solution as a 10 µ M primer solution

References

  • C. C. Richardson, Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc. Natl. Acad. Sci. USA. 54: 158-65 (1968)
  • A. M. Maxam and W. Gilbert, Methods in Enzymology, 65: 499 (1980)