Description
General description
Optimized enzyme 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.
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.
Assay Time: 100 minutes
Sample Materials
Supercoiled and linearized plasmid DNA
Supercoiled and linearized cosmid DNA
Purified PCR products
Application
For generation of highly sensitive probes for fluorescence in situ hybridization (FISH).
The Nick Translation Mix is designed for direct fluorophore-labeling of in situ probes. Fluorescein-12-dUTP and Tetramethyl-Rhodamine-5-dUTP from Roche Applied Science or other commercially available fluorophor-labeled nucleotides can be combined with the Nick Translation Mix. Direct fluorophore-labeled in situ probes are used for the detection of multi copy or very large hybridization targets on metaphase chromsomes or interphase nuclei.
For a standard labeling reaction using 1 μg template in 20 μl total reaction volume, 4 μl of 5x concentrated fluorophore labeling mix are required.
Biochem/physiol Actions
Heat inactivation: Stop the reaction by adding 1 μl 0.5 M EDTA (pH 8.0) and heating to 65 °C for 10 minutes.
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
Avoid repeated freezing and thawing.
Analysis Note
Function tested in dot spot assay.
Other Notes
Contents
1 vial with 5x concentrated solution, stabilized reaction buffer in 50% glycerol (v/v), DNA Polymerase I and DNase I.
For life science research only. Not for use in diagnostic procedures.
Denaturing of the template before nick translation is not required.
