DIG-Nick Translation Mix

Description

• General description: DIG-Nick Translation Mix is a convenient enzyme and nucleotide mixture for nick translation. It utilizes a combination of DNase and DNA Polymerase to nick one strand of the DNA helix, then incorporates labeled nucleotides as the polymerase examines, or "proofreads" the nicked site. Large plasmids, cosmids and polymerase chain reaction (PCR) fragments are all regularly used with this method and the DNA can be linearized or supercoiled. Individual templates produce consistent results in the standard 90-minutes reaction and result in an average probe length of 200 bp up to 500 bp. The molar ratio of digoxigenin (DIG)-11-deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) is adjusted to ensure that every 20th to 25th nucleotide in the newly synthesized DNA is modified with DIG. This density of haptens in the DNA yields the highest sensitivity in the immunological detection reaction.
• Specificity: Heat inactivation: Stop the reaction by adding 1μl 0.5M EDTA (pH 8.0) and heating to 65°C for 10 minutes.
• Application: DIG-Nick Translation Mix has been used for the generation of highly sensitive probes for in situ hybridization labeled with Digoxigenin-11-dUTP.[1][2][3][4]Note: The mix can also be used for filter hybridization techniques, however, for highly sensitive filter hybridization probes, we recommend that you use DIG-High Prime from Roche Applied Science. For nonradioactive labeling of in situ probes with other haptens and fluorophores, Roche Applied Science offers the Biotin-Nick Translation Mix and the Nick Translation Mix (without nucleotides).
• Quality: Function tested in a dot spot assay.
• Principle: The nick translation method is based on the ability of DNase I to introduce randomly distributed nicks into DNA at low enzyme concentrations in the presence of MgCl2.E. coli DNA Polymerase I synthesizes DNA complementary to the intact strand in a 5′→3′ direction using the 3′-OH termini of the nick as a primer. The 5′→3′ exonucleolytic activity of DNA polymerase I simultaneously removes nucleotides in the direction of synthesis. The polymerase activity sequentially replaces the removed nucleotides with isotope-labeled or hapten-labeled deoxyribonucleoside triphosphates. At low temperature (+15°C), the unlabeled DNA in the reaction is thus replaced by newly synthesized labeled DNA.
• Preparation Note: 1 vial with 5x concentrated stabilized reaction buffer in 50% glycerol (v/v) and DNA Polymerase I, DNase I, 0.25mM dATP, 0.25mM dCTP, 0.25mM dGTP, 0.17mM dTTP and 0.08mM DIG-11-dUTP (alkali-stable).Assay Time: 100 minutesSample Materials: Supercoiled and linearized plasmid DNA, Supercoiled and linearized cosmid DNA, Purified PCR products.Note: Denaturing of the template before nick translation is not required.
• Other Notes: For life science research only. Not for use in diagnostic procedures.