DIG DNA Labeling Kit

Description

• General description: DIG DNA Labeling Kit is a convenient kit for the labeling of DNA with Digoxigenin-deoxyuridine triphosphate (dUTP) using random oligonucleotides as primers. In this method, the complementary DNA strand of denatured DNA is synthesized by Klenow polymerase using the 3′-OH termini of the random oligonucleotides as primers.
• Specificity: Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or by heating to 65 °C for 10 minutes.
• Application: For Random-primed labeling of DNA probes with DIG-11-dUTP, alkali-labile. DIG-labeled DNA probes can be used for: All types of filter hybridization according to our standard protocol given in the pack insert of the special hybridization solution DIG Easy Hyb.Single-copy gene detection in total genomic DNA, even from organisms with high complexity, for example, human, barley, and wheat.[1]In situ hybridizations[2]
• Packaging: 1 kit containing 7 components.
• Quality: Function tested in a Southern blot.
• Specifications: Assay Time: Labeling: 1hour to O/NSensitivity and specificity: A single-copy human gene (tPA gene) is detected with a DIG-labeled probe in a Southern blot of 1μg digested human placenta DNA.
• Principle: DIG-labeled DNA probes are generated according to the random-primed DNA labeling method which is based on the hybridization of random oligonucleotides to the denatured DNA template. The complementary DNA strand is synthesized by Klenow enzyme which uses the 3′-OH termini of the random oligonucleotides as primers and a mixture of deoxyribonucleotides containing DIG-11-dUTP, alkali-labile for elongation. This results in incorporation of digoxigenin into the newly synthesized DNA.Note:The use of the alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization experiments with a second DIG-labeled probe.DNA probe, labeled with DIG-11-dUTP, alkali-labile must not be denatured using NaOH, but can be denatured by boiling in a waterbath.
• Other Notes: For life science research only. Not for use in diagnostic procedures.