HighFidelity ORANGE PCR Labeling Testkit

Preparation of AF594-, ATTO594- and TexasRed-labeled DNA probes by PCR

Cat. No.	Amount	Price (EUR)	Buy / Note
APP-101-ORANGE	1 kit	295,00	Add to Basket/Quote Add to Notepad

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:
AF594: λ_exc 590 nm, λ_em 617 nm, ε 92.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5) ATTO594: λ_exc 602 nm, λ_em 626 nm, ε 120.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5) TexasRed: λ_exc 583 nm, λ_em 603 nm, ε 112.0 L mmol^-1 cm^-1 (Tris-HCl pH 7.5)

Description:
HighFidelity ORANGE PCR Labeling Testkit is designed to produce randomly AF594-, ATTO594- and TexasRed-modified DNA probes by PCR to find the optimal label for the orange 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 % / 25 % 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-XX-AF594
1x 10 μl (1 mM)

dUTP-XX-ATTO-594
1x 10 μl (1 mM)

dUTP-TexasRed
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 % dUTP-XX-AF594 & dUTP-XX-594 or with 25 % dUTP-TexasRed 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.

50 % substitution

Component	Volume	Final concenctration
PCR-grade water	X μl
High Fidelity Labeling Buffer (10x)	2 μl	1x
1 mM dATP/dCTP/ dGTP working solution (s. 1.1)	2 μl	100 μM
1 mM dTTP working solution (s. 1.2)	1 μl	50 μM
1 mM labeled-dUTP	1 μl	50 μM
forward primer (10 μM)	X μl	0.1 - 1 μM (e.g. 0.3 μM 500 bp forward primer)
reverse primer (10 μM)	X μl	0.1 - 1 μM (e.g. 0.3 μM 500 bp reverse primer)
template DNA	X μl	1 - 10 ng genomic DNA (e.g. 1 ng Lambda DNA)
High Fidelity Polymerase (2.5 units/μl)	1 μl	2.5 units
Total volume	20 μl

25 % substitution

Component	Volume	Final concenctration
PCR-grade water	X μl
High Fidelity Labeling Buffer (10x)	2 μl	1x
1 mM dATP/dCTP/ dGTP working solution (s. 1.1)	2 μl	100 μM
1 mM dTTP working solution (s. 1.2)	1.5 μl	50 μM
1 mM labeled-dUTP	0.5 μl	50 μM
forward primer (10 μM)	X μl	0.1 - 1 μM (e.g. 0.3 μM 500 bp forward primer)
reverse primer (10 μM)	X μl	0.1 - 1 μM (e.g. 0.3 μM 500 bp reverse primer)
template DNA	X μl	1 - 10 ng genomic DNA (e.g. 1 ng Lambda DNA)
High Fidelity Polymerase (2.5 units/μl)	1 μl	2.5 units
Total volume	20 μl

Recommended cycling conditions

Cycle step	Temperature	Time	Cycles
Initial denaturation	95°C	2 min	1x
Denaturation Annealing¹⁾ Elongation²⁾	95°C 58°C 68°C	20 sec 30 sec 60 sec	30x
Final Elongation	68°C	2 min	1x

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:

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HighFidelity ORANGE PCR Labeling Testkit

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