Uses
DIG-High Prime DNA Labeling and Detection Starter Kit I has been used in a variety of hybridization techniques:
- in Southern blots
- in northern blots
- in dot blots
- in colony and plaque hybridizations
- for all types of filter hybridization
- for single-copy gene detection in total genomic DNA, even from organisms with high complexity, for example, human, barley and wheat
DIG-labeled probes are generated at high yield within one hour or after overnight incubation.
General Description
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. This kit contains a ready-made blocking solution, combined stock solution of of nitroblue tetrazolium (NBT)/ 5-bromo-4-chloro-3-indolyl-phosphate (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. The sample material can be: DNA fragments of at least 100bp, linearized plasmid, cosmid or λDNA, or supercoiled DNA. 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 (10ng) with this method. In 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. This is a convenient kit for random-primed labeling of DNA templates with DIG-11-dUTP, alkali-labile, and color detection of the DIG-labeled hybrids. This product is designed as a safer chemical.? The DIG System was established as a sensitive and cost-effective alternative to radioactivity for the labeling and detection of nucleic acids. There are many available publications that prove the versatility of the DIG System, so use of radio-labeling is no longer the only option for labeling of DNA for hybridization.
