HighFidelity Biotin16 PCR Labeling Kit

For general laboratory use.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Description:
HighFidelity Biotin16 PCR Labeling Kit is designed to produce randomly Biotin16-modified DNA probes by PCR. Such probes are ideally suited for in situ hybridization and Northern Blot experiments. PCR-based labeling is superior to random-primed labeling by Klenow fragment if template amounts are limited or amplification of a specific DNA fragments is required.

Biotin-16-dUTP is efficiently incorporated into DNA as substitute for its natural counterpart dTTP using an optimized reaction buffer and a High Fidelity Polymerase. 50 % Biotin-16-dUTP substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of Biotin-16-dUTP/dTTP ratio however, can easily be achieved with the single nucleotide format. The resulting Biotin16-modified DNA probe can subsequently be detected by HRP- or AP-modified Streptavidin.

The kit contains sufficient reagents for 175 labeling reactions of 20 μl each (50 % Biotin16-dUTP substitution, 100 μM dATP/dGTP/dCTP, 50 μM dTTP, 50 μM Biotin16-dUTP).

Content:
High Fidelity Polymerase
in storage buffer with 50% glycerol (v/v)
1x 200 μl (500 units, 2.5 units/μl)

High Fidelity Labeling Buffer
1x 500 μl (10x)

dATP - Solution
1x 20 μl (100 mM)

dGTP - Solution
1x 20 μl (100 mM)

dCTP - Solution
1x 20 μl (100 mM)

dTTP - Solution
1x 20 μl (100 mM)

Biotin-16-dUTP
1x 200 μl (1 mM)

Lambda DNA
1x 20 μl (100 ng/μl)

500 bp forward primer
1x 20 μl (10 μM)

500 bp reverse primer
1x 20 μl (10 μM)

PCR-grade water
1x 1.2 ml

To be provided by user
DNA template
Primer
DNA purification tools (optional)

1. Preparation of working solutions

1.1 Preparation of 1 mM dATP/dCTP/dGTP working solution

• Thaw 100 mM dATP, 100 mM dCTP and 100 mM dGTP solutions on ice, voretex and spin-down briefly.
• Prepare a 1:100 dilution with PCR-grade water to achieve a final concentration of 1 mM (e.g. 2 μl 100 mM dATP + 2 μl 100 mM dCTP + 2 μl 100 mM dGTP + 194 μl PCR-grade water).
• 1 mM ATP/CTP/GTP working solution can be stored at -20°C. Prepare aliquots to avoid freeze/thaw cycles.

1.2 Preparation of 1 mM dTTP working solution

• Thaw 100 mM dTTP solution on ice, voretex and spin-down briefly.
• Prepare a 1:100 dilution with PCR-grade water to achieve a final concentration of 1 mM (e.g. 2 μl 100 mM dTTP + 198 μl PCR-grade water).
• 1 mM dTTP working solution can be stored at -20 °C. Prepare aliquots to avoid freeze/thaw cycles.

3. Standard PCR Labeling protocol

The standard protocol is set-up for labeling of a 500 bp DNA fragment. An optimal balance between reaction and labeling efficiency is typically achieved with 50% Biotin-16-dUTP substitution following the standard protocol below however, individual optimization might improve results for individual applications.

• Assemble the PCR on ice in the order stated below (DNAse-free reaction tube).
• Voretex and spin-down briefly.

Recommended cycling conditions

¹⁾The annealing temperature depends on the melting temperature of primers used.
²⁾The elongation time depends on the length of fragments to be amplified. A time of 2 min/kbp is recommended. Elongation at 72°C works as well.

For optimal amplification results and high incorporation rates an individual optimization of the recommended PCR assay and cycling conditions may be necessary for each new primer-template pair.

4. Probe purification:

Probe purification is not required for most hybridization experiments. If a downstream application requires purification (e.g. concentration determination by absorbance measurement) we recommend silica-membrane or gel filtration-based purification.

Related products: Biotin-16-dUTP, #NU-803-BIO16

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