For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Biotin14 Nick Translation Labeling Kit contains all reagents (except template and materials for purification of the probe) required for nick translation-based Biotin labeling of DNA providing a highly efficient, easy-to-perform and rapid labeling technology. The labeling principle is similar to the BioNickTM DNA Labeling system (ThermoFisher Scientific).
Nick translation labeling is based on the reverse activities of Polymerase I and DNase I. DNase I is able to introduce randomly distributed nicks to double stranded DNA at low enzyme concentrations. The 5'→3' exonuclease activitiy of Polymerase I removes nucleotides from the 3' side of the nick while synthesizing a partial new complementary strand using the 3'-OH termini as primer. In the presence of labeled dUTP Polymerase I incorporates labeled dUTP instead of dTTP.
The BIO14 NT labeling mix contains an optimized Biotin-labeled dATP for incorporation into DNA by nick translation using DNA Polymerase I.
Biotin-14-dATP is enzymatically incorporated into DNA as substitute for its natural counterpart dATP. Optimal substrate properties and thus labeling efficiency as well as an efficient detection of the Biotin moiety is ensured by a 14-atom linker attached to the N6 position of adenine.
The well balanced polymerase / nuclease activities of the enzyme mix ensure the generation of highly labeled double stranded DNA fragments.
The resultant Biotin-labeled DNA is suitable for application in FISH and other nucleic acid hybridization assays.
Enzyme mix (red cap)
2 units/μl Polymerase I, 0.02 units/μl DNase I in storage buffer
10x NT labeling buffer (green cap)
10x BIO14 NT labeling mix (purple cap)
0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.25 mM dATP, 0.25 mM Biotin-14-dATP, pH 7.5
Stop buffer (yellow cap)
0.5 M EDTA, pH 8.0
PCR-grade water (white cap)
Recommended NT assay (General Protocol):
Sample material can be supercoiled or linearized plasmid DNA, cosmid or BAC DNA, whole or partial chromosomes or purified PCR products. PCR products should be purified by silica membrane adsorption to remove unincorporated dNTPs (e.g. PCR Purification Kit, #PP-201).
Prepare the following reaction mixture in a sterile vial.
20 μl Nick translation labeling assay
|2 μl||10x NT labeling buffer||green cap|
|2 μl||10x BIO14 NT labeling mix||purple cap|
|1-1.5 μg||template DNA||-|
|2 μl||10x Enzyme mix||red cap|
|Fill up to 20 μl||PCR-grade water||white cap|
Purification of the probe:
Some downstream applications require the removal of unincorporated dNTPs (labelled and unlabelled). To remove unincorporated nucleotides from the reaction mixture prior to its use in subsequent experiments one of the following procedures is recommended:
1. Purification by silica-gel membrane adsorption - PCR Purification Kit, #PP-201
The Jena Bioscience PCR Purification Kit provides a simple and efficient way to purify DNA fragments larger than 100 bp. The preparation is based on a silica-membrane technology for binding DNA in high-salt and elution in low-salt buffer. Please refer to the instruction manual.
2. Purification by Isopropanol precipitation
Add 1μl glycogene (2 mg/ml), 2 μl sodium acetate (3 M) and 14 μl isopropanol to the reaction mixture and mix well but gently. Incubate on RT for 15 min and spin down at maximum speed at 4°C for 30 min. Discard the supernatant and wash 2x with 70% ethanol (spin down at maximum speed for 5 min).
3. Purification by Centrifugal Filter Units
Unincorporated nucleotides can be removed by centrifugation using centrifugal filter units. Select the filter unit by its cut-off for DNA fragments and follow the manufacturer's instructions.