Description
Source: E. coli strain containing a modified clone of T4 Polynucleotide Kinase.
Description:
OptiKinase™ is a modified version of T4 Polynucleotide Kinase (PNK)(1,2) which provides greater 32P or 33P labeling efficiency of single-stranded oligonucleotides. Unlike native
T4 PNK which exhibits a bias against the efficient labeling of certain oligonucleotides, particularly those with 5'-C ends(3), OptiKinase exhibits little or no such discrimination. OptiKinase, like the unmodified native T4 PNK enzyme, catalyzes the transfer of the terminal phosphate of ATP to 5' hydroxyl termini of polynucleotides such as DNA and RNA. Use of OptiKinase allows for higher levels of phosphorylation with greater consistency.
Tested User Friendly
Properties:
Molecular Weight: 140,000 (4 x 33,000) Da
Optimum pH: 7.6 (Tris-HCI)
Optimum Temperature: 37°C
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating exonucleases, endonucleases and ribonucleases.
Storage Buffer:
50mM Tris-HCl (pH 7.5), 1.0mM DTT, 0.1mM EDTA, 50% glycerol.
Shipping and Storage:
Shipped on dry ice. Store at -20°C.
Assay Conditions:
The reaction mixture (250 µl) contains 50mM Tris-HCI (pH 7.6), 100µM radiolabeled ATP, 10mM MgCl2, 10mM 2-mercaptoethanol, 20µM spermidine and substrate. Incubation is at
37°C.
Unit Definition:
One unit is the amount of enzyme required to incorporate 1 nmol of phosphate from radiolabeled ATP into DNA substrate in 30 min at 37°C.
Concentration:
10 units/µl
Tested User FriendlyTM Functional Test:
Phosphorylation of oligonucleotide is accomplished with > 60% utilization of radioactive ATP.
Applications:
*5'-labeling of single-stranded oligonucleotides.
*Phosphorylation of DNA and RNA.
5'-End Labeling Protocol
*This protocol is written for labeling 5 pmol of oligonucleotide in water or buffer. The reaction may be scaled up or down as needed.
For a standard reaction, combine the following:
Oligonucleotide (5 pmol) __ µl
OptiKinase Reaction Buffer (10X) 2.5 µl
[Gamma-32P]ATP* __ µl
Water __ µl
OptiKinase 10 units
––––––––
Total 25 µl
*Mix the contents well and centrifuge briefly. Incubate at 37°C for 30 minutes.
*Terminate the reaction by heating at 65°C for 10 minutes. Proceed with the separation of the 5'-end-labeled oligonucleotide from precursor ATP by thin-layer or column chromatography according to standard methods.
* Either [γ-32P]ATP or [γ-33P]ATP ( greater than or equal to 3,000 Ci/mmol) may be used. The number of pmol of radioactive ATP should be at least equimolar with the number of pmol of
5'-ends. In contrast to wild-type T4 PNK, OptiKinase can accomplish efficient labeling without the need for excess radioactive ATP.
Non-Isotopic Phosphorylation
To make unlabeled phosphorylated substrate, replace radioactive ATP with cold ATP to 1mM final concentration.
References:
*RICHARDSON, C. C. (1981) The Enzymes, 3rd edition, ed. P.D. Boyer, (Academic Press, New York) 14, 299-314.
*MAXAM, A. M. AND GILBERT, W. (1980) Methods in Enzymology 65, 499-560.
*VAN HOUTEN, V., DENKERS, F., VAN DIJK, M., VAN DEN BREKEL, M. AND BRAKENHOFF, R. (1998) Anal. Biochemistry 265, 386-389.
*Tech Tip 205, USB OptiKinase Eliminates Base Bias in Labeling of Oligonucleotide
5'-Ends, USB Corporation, Cleveland, Ohio.
Functionally Tested 10X OptiKinase Reaction Buffer (1 ml included, PN 78336):
0.5M Tris-HCI (pH 7.5), 100mM MgCl2, 50mM DTT.