Not available outside of the UK & Ireland.

Analysis Note

Specific Activity: The standard assay routinely yields a specific activity of 2 x 109 dpm/µg, using different substrate DNAs after 10 minutes of incubation.Assay Time: 30 minutesSample Materials DNA fragments of at least 100 bp Linearized plasmid, cosmid or λDNA Supercoiled DNA Or minimal amounts of DNA (10 ng), e.g., DNA restriction fragments isolated from gels or in molten agaroseNote: The length of the DNA to be labeled does not influence the reaction. Maximal incorporation may require a prolonged incubation period of 30 to 60 minutes.

Application

For the labelling of DNA with radioactive dCTP using random oligonucleotides as primers.High Prime-labelled probes are used in a variety of hybridization techniques: Southern blotsNorthern blotsDot/slot blotsScreening of gene librariesIn situ hybridizationsHigh Prime has been used to label the probes by random hexamer labelling and labelling of probes with ³²P by random priming.

General description

Enzyme and nucleotide mixture for rapid random-primed labeling of DNA with [³²P], [³⁵S], or [³H] dCTP. In this method, the complementary DNA strand of denatured DNA is synthesized by Klenow polymerase using the 3′-OH termini of the random oligonucleotides as primers.

High Prime, a novel labeling mixture, contains optimal concentrations of nucleotides and primers in a highly efficient reaction mix with Klenow enzyme. This convenient "all-in-one" principle of High Prime reduces pipetting steps to a minimum, and increases accuracy and reproducibility of labeling reactions.

High Prime is used for rapid random-primed labeling of DNA with [³²P], [³⁵S], or [³H]dCTP to a guaranteed specific activity of >2 x 10⁹ dpm/µg. Incubation time is only 10 minutes at +37°C.High Prime guarantees efficient labeling of: DNA amounts ranging from 10 ng to 3 µg in a standard reaction DNA of different lengths ranging from small restriction fragments to l or cosmid DNA DNA, supercoiled or linearized DNA in low melting-point agarose.High Prime-labeled DNA probes are suitable for single-copy gene detection in mammalian DNA in Southern, northern, and dot blots, as well as in colony- or plaque hybridization.

Contents:5x concentrated random primer mix: 1 U/µl Klenow polymerase, labeling grade, 0.125 mM dATP, 0.125 mM dGTP, 0.125 mM dTTP in 50% (v/v) glycerol.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Principle

Labeled probes are generated with High Prime according to the random-primed labeling technique. High Prime is a specifically developed reaction mixture containing random oligonucleotides, Klenow polymerase, labeling grade, dATP, dGTP, and dTTP in an optimized reaction buffer concentrate in 50% glycerol for rapid and efficient labeling of DNA with [³²P]-, [³⁵S]-, or [³H]-labeled dCTP.

Quality

Function tested in the standard assay.

Specificity

Heat inactivation: Stop the reaction by adding 2 µl 0.2 M EDTA (pH 8.0) and/or by heating to 65 °C for 10 minutes.