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For Oligonucleotide Labeling
DNA synthesizer
Standard reagents for oligonucleotide synthesis (tetrazol, etc.)
Absolute, amine-free dimethylformamide (DMF)
5'-Amino modifier
3'-Phosphate CPG support
Standard phosphoramidites
0.1 M sodium borate buffer (pH 8.5)
For Ethanol Precipitation of Oligonucleotide
3 M sodium acetate buffer (pH 8.5)
Ice-cold absolute ethanol
For Oligonucleotide Purification by HPLC
HPLC
Vacuum centrifuge
POROS OligoR3 separation medium (PerSeptive Biosystems, Inc., 4.6 × 50 mm column). This separation medium is recommended to obtain optimal purification results.
100 mM Triethylammoniumacetate (pH 6.9)
Acetonitrile
Do not use Reversed Phase (RP) separation media because labeled oligonucleotides tend to stick irreversibly to the RP-material, resulting in lower yields of labeled oligonucleotides.
For Quality Control of HPLC-Purified Oligonucleotides
LightCycler® Red 640 shows an excitation maximum at 625 nm and an emission maximum at 640 nm (in 2 mM Tris-buffer, pH 8.3). Fig. 1: Emission spectra of fluorescent dyes.
Labeling Principle
5'-amino-substituted-3'-phosphorylated oligonucleotides react with the LightCycler® Red 640-NHS ester in a sodium borate buffer/dimethylformamide (DMF) mixture at pH 8.5 - 9.0.",
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Vial/Bottle
Label
Function/Description
Content
1
LightCycler® Red 640 NHS-ester for ≥250 nmol oligonuc.
Contains a sufficient amount of LightCycler® Red 640-NHS ester for labeling a minimum of 5 × 50 nmol oligonucleotides.