High Prime

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.

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 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.

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.

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.

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 blots

• Northern blots
• Dot/slot blots
• Screening of gene libraries
• In situ hybridizations

High Prime has been used to label the probes by random hexamer labelling and labelling of probes with ³²P by random priming.

Function tested in the standard assay.

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.

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 minutes
Sample 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 agarose

Note: 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.

仅用于生命科学研究。不可用于诊断。