Non disponible en dehors du Royaume-Uni et de l'Irlande

Application

For generation of highly sensitive probes for in situ hybridization labeled with Biotin-16-dUTP.Note: The mix can also be used for filter hybridization techniques, however, for highly sensitive filter hybridization probes, we recommend that you use Biotin-High Prime from Roche Applied Science.For nonradioactive labeling of in situ probes with other haptens, Roche Applied Science offers the DIG-Nick Translation Mix or the Nick Translation Mix.

General description

Convenient enzyme and nucleotide mixture.Nick translation 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. The molar ratio of Biotin-16-dUTP to dTTP is adjusted to ensure that every 20^th to 25^th nucleotide in the newly synthesized DNA is modified with biotin. This density of haptens in the DNA yields the highest sensitivity in the immunological detection reaction.Large plasmids, cosmids, and 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 base pairs up to 500 base pairs.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Physical form

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 Biotin-16-dUTP.

Preparation Note

Assay Time: 100 minutesSample Materials Supercoiled and linearized plasmid DNASupercoiled and linearized cosmid DNAPurified PCR productsNote: Denaturing of the template before nick translation is not required.

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.

Quality

Function tested in a dot blot assay.

Specificity

Heat inactivation: Stop the reaction by adding 1 µl 0.5 M EDTA (pH 8.0) and heating to 65 °C for 10 minutes.