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.
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.
Sensitivity and specificity: Standard assay uses 1 µg of linearized pBR328. It is usual to expect 0.8 µg labeled DNA after 1 h of labeling, and 2.3 µg labeled DNA after 20 h 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.
- DIG-High Prime, 5x conc., 50 µl
- DIG-labeled Control DNA, 20 µl, (5 µg/ml) pBR328 (linearized with Bam HI)
- DNA Dilution Buffer, 3 x 1 ml
- Anti-digoxigenin-AP Conjugate, 50 µl
- CSPD, ready-to-use, 50 ml
- Blocking Solution, 10x conc., 4 x 100 ml
- DIG Easy Hyb Granules, 4 x 100 ml