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Random Primer DNA Labeling Kit
Premixed solution for the labeling of DNA with radiolabeled dCTP using random sequence oligonucleotides.
The method is based on the hybridization of oligonucleotides of all possible sequences to the denatured template DNA to be labeled. The cDNA strand is synthesized by a "klenow" fragment of DNA Polymerase I, using random oligoncleotides as primers. By substituting a radiolabeled nucleotide for a non-radioactive equivalent in the reaction mixture, the newly synthesized cDNA is made radioactive. The labeling mix system is a specially developed reaction mixture for enhanced convenience and performance. The reaction mixture contains random olionucleotides, a Klenow fragment of DNA Polymerase I, dATP, dGTP, dTTP, and a reaction buffer concentrate. The DNA labeling mix allows the labeling of the template DNA to a specific activity after only 10 minutes of incubation. This rapid labeling is accomplished with the use of the Klenow fragment, which lacks 5'-3' exonuclease activity, and by the use of nonamer primers giving more efficient priming from the template at 37ºC. The labeling mix method enables the labeling of small amounts of DNA (10-20ng), such as restriction fragments isolated from gels. Fragments can be labeled directly in low melting temperature agarose gel slices. The labeled probes are used in various hybridization techniques, such as Southern and Northern blots, in-situ hybridization and screening of gene libraries.
Kit Reagents
· 1
vial containing 100µl DNA labeling mixture. Each vial is sufficient for 25
labeling assays.
Storage and Stability
· The premixed solution should be stored at -20ºC. Avoid repeated changes in the solution temperature.