HighFidelity RED PCR Labeling Testkit

For general laboratory use.

Shipping: shipped on gel packs

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

Shelf Life: 12 months

Spectroscopic Properties:
Cy5: λ_exc 649 nm, λ_em 670 nm, ε 250.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5) AF647: λ_exc 648 nm, λ_em 671 nm, ε 270.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5) ATTO643: λ_exc 643 nm, λ_em 665 nm, ε 150.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5)

Description:
HighFidelity RED PCR Labeling Testkit is designed to produce randomly Cy5-, AF647- and ATTO643-modified DNA probes by PCR to find the optimal label for the red emission wavelength range. Such probes are ideally suited for Fluorescence in situ hybridization (FISH) and Northern Blot experiments. PCR-based labeling is superior to random-primed labeling with Klenow fragment if template amounts are limited or amplification of a specific DNA fragments is required. Amplification of probes up to 4kbp is feasible.

All labeled-dUTP are efficiently incorporated into DNA as substitute for its natural counterpart dTTP using an optimized reaction buffer and a High Fidelity Polymerase blend consisting of Taq polymerase and a proofreading enzyme. 50 % labeled-dUTP substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of labeled-dUTP/dTTP ratio however, can easily be achieved with the single nucleotide format.

Content:
High Fidelity Polymerase
in storage buffer with 50% glycerol (v/v)
1x 40 μl (100 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)

dUTP-Cy5
1x 10 μl (1 mM)

dUTP-XX-AF647
1x 10 μl (1 mM)

dUTP-ATTO-643
1x 10 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% labeled-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.
• Perform assay set-up and reaction under low-light conditions.

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:

• Aminoallyl-dUTP-Cy5, #NU-803-Cy5
• Aminoallyl-dUTP-XX-AF647, #NU-803-XX-AF647
• Aminoallyl-dUTP-ATTO-643, #NU-803-643

BIOZ Product Citations: