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HighFidelity ATTO488 PCR Labeling Kit

Preparation of ATTO488-labeled DNA probes by PCR

Cat. No. Amount Price (EUR) Buy / Note
APP-101-488-S 10 reactions x 20 μl 145,00 Add to Basket/Quote Add to Notepad
APP-101-488-L 50 reactions x 20 μl 485,00 Add to Basket/Quote Add to Notepad
Structural formula of HighFidelity ATTO488 PCR Labeling Kit (Preparation of ATTO488-labeled DNA probes by PCR)
Structural formula of HighFidelity ATTO488 PCR Labeling Kit
excitation and emission spectrum of ATTO 488
excitation and emission spectrum of ATTO 488

For in vitro use only!

Shipping: shipped on blue ice

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

Shelf Life: 12 months

Spectroscopic Properties: λexc 500 nm, λem 520 nm, ε 90.0 L mmol-1 cm-1 (Tris-HCl pH 7.5)

Description:
HighFidelity ATTO488 PCR Labeling Kit is designed to produce randomly ATTO488-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-ATTO-488 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-ATTO-488 substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of dUTP-XX-ATTO-488/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-ATTO-488 substitution, 100 μM dATP/dGTP/dCTP, 50 μM dTTP, 50 μM dUTP-XX-ATTO-488).

Content:
High Fidelity Polymerase
in storage buffer with 50% glycerol (v/v)
#APP-101-488-S: 1x 40 μl (100 units, 2.5 units/μl)
#APP-101-488-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-ATTO-488
#APP-101-488-S: 1x 10 μl (1 mM)
#APP-101-488-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-ATTO-488 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.


ComponentVolumeFinal concenctration
PCR-grade waterX μl
High Fidelity Labeling Buffer (10x)2 μl1x
1 mM dATP/dCTP/ dGTP working solution (s. 1.1)2 μl100 μM
1 mM dTTP working solution (s. 1.2)1 μl50 μM
1 mM dUTP-XX-ATTO-4881 μl50 μM
forward primer
(10 μM)
X μl0.1 - 1 μM (e.g. 0.3 μM 500 bp forward primer)
reverse primer
(10 μM)
X μl0.1 - 1 μM (e.g. 0.3 μM 500 bp reverse primer)
template DNAX μl1 - 10 ng genomic DNA (e.g. 1 ng Lambda DNA)
High Fidelity Polymerase (2.5 units/μl)1 μl2.5 units
Total volume20 μl

Recommended cycling conditions

Cycle stepTemperatureTimeCycles
Initial
denaturation
95°C2 min1x
Denaturation
Annealing1)
Elongation2)
95°C
58°C
68°C
20 sec
30 sec
60 sec
30x
Final
Elongation
68°C2 min1x

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-ATTO-488, #NU-803-XX-488