Convenient kit for random-primed labeling of DNA templates with DIG-11-dUTP, alkali-labile, and color detection of the DIG-labeled hybrids. 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.This kit was assembled with convenience in mind, offering a ready-made blocking solution, combined stock solution of NBT/BCIP, and DIG Easy Hyb granules. The DIG-High Prime mixture includes stabilized Klenow Enzyme, nucleotides, primers, and reaction buffer, all in one convenient reagent.
Sample Materials
The DIG High Prime DNA Labeling and Detection Starter Kit I uses digoxigenin (DIG), a steroid hapten, to label DNA probes for hybridization and subsequent color detection by enzyme immunoassay. The "random primed" DNA labeling method originally developed by Feinberg and Vogelstein is based on the hybridization of oligonucleotides of all possible sequences to the denatured DNA to be labeled. The input DNA serves as a template for the synthesis of labeled DNA, and is not degraded during the reaction, making it possible to label minimal amounts of DNA (10 ng) with this method. The complementary DNA strand is synthesized by Klenow polymerase using the 3'-OH termini of the random oligonucleotides as primers. Modified deoxyribonucleoside triphosphates, labeled with digoxigenin present in the reaction are incorporated into the newly synthesized complementary DNA strand.
Function tested in a dot blot.
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