HighFidelity Cy3 PCR Labeling Kit

For research use only.

Description:
HighFidelity Cy3 PCR Labeling Kit is designed to produce randomly Cy3-modified DNA probes by PCR. 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.

dUTP-XX-Cy3 is 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 % dUTP-XX-Cy3 substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of dUTP-XX-Cy3/dTTP ratio however, can easily be achieved with the single nucleotide format.

The kit contains sufficient reagents for 10 labeling reactions (S-Pack) or 50 labeling reactions (L-Pack) of 20 μl each (50% dUTP-XX-Cy3 substitution, 100 μM dATP/dGTP/dCTP, 50 μM dTTP, 50 μM dUTP-XX-Cy3).

Shipping: shipped on gel packs

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

Shelf Life: 12 months

Spectroscopic Properties: λ_exc 550 nm, λ_em 570 nm, ε 150.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5)

Content:
High Fidelity Polymerase
in storage buffer with 50% glycerol (v/v)
#APP-101-Cy3-S: 1x 40 μl (100 units, 2.5 units/μl)
#APP-101-Cy3-L: 2x 40 μl (2x 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-XX-Cy3
#APP-101-Cy3-S: 1x 10 μl (1 mM)
#APP-101-Cy3-L: 5x 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% dUTP-XX-Cy3 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-XX-Cy3, #NU-803-XX-Cy3