DIG-High Prime DNA Labeling and Detection Starter Kit II
For 12 random-primed DNA labeling reactions with DIG-11-dUTP, alkali-labile, and 24 chemiluminescence detection reactions with CSPD, ready-to-use.
Product No. 11585614910
Pack Size 1 kit for up to 12 labeling reactions and detection of 24 blots, 10 ng to 3 μg per assay, blots of 100 cm2
Overview：
The High Prime method of labeling offers many benefits: in addition to the optimized labeling efficiency, plasmid, cosmid, and phage DNA, linearized or supercoiled can be used as templates, yielding the same labeling efficiency.
Contents
1. DIG-High Prime, 5x concentrated
2. DIG-labeled Control DNA (5 μg/ml), pBR328 (linearized with Bam HI)
3. DNA Dilution Buffer
4. Anti-digoxigenin-AP Conjugate
5. CSPD, ready-to-use
6. Blocking Solution, 10x concentrated
7. DIG Easy Hyb Granules
Application
DIG-High Prime labeled probes can be used in northern, Southern-, and dot-blot analysis, as well as colony and plaque hybridizations. This kit offers random-primed labeling of DNA templates with DIG-11-dUTP, alkali-labile, and chemiluminescent detection of the DIG-labeled hybrids. This kit was assembled with convenience in mind, offering ready-to-use CSPD supplied with a dripping device for easy application, ready-made blocking solution, and DIG Easy Hyb granules. The DIG-High Prime mixture includes stabilized Klenow enzyme, nucleotides, primers, and reaction buffer, all in one convenient reagent.
Details
Sample Materials
DNA fragments of at least 100 bp
Linearized plasmid, cosmid or λDNA
Supercoiled DNA
Sensitivity and specificity: Standard assay uses 1 μg of linearized pBR328. It is usual to expect 0.8 μg labeled DNA after 1 hour of labeling, and 2.3 μg labeled DNA after 20 hours reaction. In a dot blot hybridization, 0.03 pg of homologous DNA is detectable with chemiluminescence (probe concentrations at 20 ng/ml). A single copy human gene (t-PA) is detectable in a Southern blot, loading 1 μg of digested placenta DNA.
Principle
The DIG High Prime DNA Labeling and Detection Starter Kit II uses digoxigenin (DIG), a steroid hapten, to label DNA probes for hybridization and subsequent chemiluminescence 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 solely 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.
Quality
Function tested in a dot blot.