HEXANUCLEOTIDE MIX

General description

Convenient oligonucleotide mixture for rapid random-primed labeling of DNA with radioactive, digoxigenin- or biotin-labeled nucleotides. In this method, the complementary DNA strand 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

Hexanucleotide Mix is a mixture of hexanucleotides of all possible sequences for random-primed DNA labeling.
Labeled DNA probes with high specific activity are used in a variety of hybridization techniques:

• Screening of gene libraries
• Southern and northern blots
• In situ hybridizations
• RT-PCR
• Generation of cDNA libraries
• Synthesis of first-strand cDNA
• in the determination of vector titer
• Second strand synthesis

Features and Benefits

The product is a 10x concentrated mixture of random hexanucleotides. Statistically, the mix may contain up to 4,096 different hexanucleotides, but these are probably present in differing amounts.

Contents
10x concentrated mixture of hexanucleotides (62.5 A₂₆₀ units/ml) in reaction buffer [0.5M Tris- HCl, 0.1M MgCl₂, 1mM dithioerythritol (DTE), 2mg/ml BSA, pH 7.2 (+20°C)]
Note: The mix is identical to that supplied in vial 5 of the DIG DNA Labeling and Detection Kit and of the DIG DNA Labeling Kit and in vial 6 of the Random Primed DNA Labeling Kit.

Quality

Function tested in the Random Primed DNA Labeling Kit.

Principle

The "random primed" DNA labeling method developed by Feinberg and Vogelstein is based on the hybridization of a mixture of all possible hexanucleotides to the DNA to be labeled. All sequence combinations are represented in the hexanucleotide primer mixture, which leads to binding of primer to the template DNA in a statistical manner. Thus, an equal degree of labeling along the entire length of the template DNA is guaranteed. The complementary strand is synthesized from the 3′-OH termini of the random hexanucleotide primer using Klenow enzyme, labeling grade. Modified deoxyribonucleoside triphosphates ([³²P], [³⁵S],[³H], or [¹²⁵I] digoxigenin- or biotin-labeled) present in the reaction are incorporated into the newly synthesized complementary DNA strand.

Preparation Note

Assay Time
Standard labeling (radioactive): 50 minutes
Labeling assay with digoxigenin-11-dUTP: 80 minutes

Sample Materials

• DNA fragments
• Linearized plasmid DNA
• λDNA

Note: The length of the DNA fragments to be labeled does not influence the reaction. DNA fragments of 100bp length are labeled equally well as linearized plasmid- or λ-DNA. The input DNA serves solely as template for the synthesis of labeled DNA, and is not degraded during the reaction, making it possible to label minimal amounts of DNA (10ng) using this method.

Synthesis: All 4 bases are used to synthesize this random hexanucleotide mix. In the initial reaction, starter nucleotides are linked to a solid phase support. In subsequent coupling reactions, equimolar amounts of the 4 dNTPs are linked to the starter nucleotides until hexamers are generated. The hexamers are then released from the solid phase support.
Post-synthesis: The oligonucleotides are HPLC purified, desalted, and 5′-phosphorylated.

Analysis Note

Absorption: 62.5 A₂₆₀ units correspond to 2.5 mg/ml of hexanucleotides.

Other Notes

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