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.
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.
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.
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:
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.
- 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.
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.