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BioNick™ DNA Labeling System (Invitrogen™)

The BioNick™ DNA Labeling System is ideal for generating biotinylated DNA probes by nick translation optimized for use in non-radioactive in situ hybridizations. These probes can also be used in Southern or northern blots, plaque lifts, colony hybridizations, and dot blot hybridizations. Using the BioNick™ DNA Labeling System:

• DNA is labeled with biotin-14-dATP, producing probe sizes from 50 to 500 bp
• One reaction labels 1 µg of template DNA

Random Primers DNA Labeling System (Invitrogen™)

The Random Primers DNA Labeling System is ideal for radioactively labeling DNA, particularly fragments <1 kb. The Random Primers DNA Labeling System:
––Yields >109 cpm/µg control DNA using [ α-32 P]-dCTP
–25 ng of DNA in one reaction


Performance and Quality Testing: Incorporation of a radioactively labeled nucleotide is verified using control DNA in a random primers labeling reaction.

SuperScript™ Indirect cDNA Labeling System (Invitrogen™)

The SuperScript® Indirect cDNA Labeling System is an array labeling kit based on proven methods of indirect cDNA labeling. The optimized system provides:

• SuperScript® III RT for generating high cDNA yields
• A proprietary nucleotide mixture to increase signal intensity
• A convenient kit format that saves valuable time

In this indirect labeling method, mRNA or total RNA is reverse transcribed using SuperScript® III RT, incorporating amino-modified dUTP and dATP into the synthesized cDNA. The template RNA is then degraded by base hydrolysis, and the reaction is neutralized with acid. The amino-modified cDNA is then purified to remove unincorporated nucleotides, primers, and buffers. In the second step, the modified cDNA is coupled with the active form of a fluorescent dye. The fluorescently labeled cDNA is purified with a S.N.A.P.™ spin column to remove any unreacted dye. The resulting fluorescently labeled cDNA is ready for hybridization to microarrays (Figure 1).

The SuperScript® Indirect cDNA Labeling System provides a complete system that includes both the core kit reagents (except dyes) and a purification module. The SuperScript® Indirect cDNA Labeling Module includes just the core kit reagents. Both provide the flexibility to use fluorescent nucleotides of your choice.

SuperScript™ Direct cDNA Labeling Module, no purification (Invitrogen™)

The SuperScript® Direct cDNA Labeling System is a microarray labeling kit that combines the performance of SuperScript® III Reverse Transcriptase (RT) with the simplicity of direct labeling to yield highly fluorescent labeled cDNA that accurately represents your initial mRNA sample. In this method (Figure 1), anchored oligo(dT) anneals to the mRNA template. SuperScript® III RT extends from the priming site, incorporating fluorescent dNTPs directly to produce labeled cDNA. Template RNA is degraded by base hydrolysis. Free nucleotides and other contaminants are removed using purification columns before hybridization to a microarray.

The SuperScript® Direct cDNA Labeling System uses 800 units of SuperScript® III RT to generate:

• High cDNA yields without an additional enzyme "spike" (Figure 2)
• Strong signal intensities (Figure 3)
• A fast and simple protocol that minimizes "hands on" time and provides superior reproducibility

Biotin DecaLabel DNA Labeling Kit (Thermo Scientific™)

Thermo Scientific Biotin DecaLabel DNA Labeling Kit is an advanced system for the efficient synthesis of biotin-labeled DNA probes, based on an improved random-primed labeling method developed by Feinberg and Vogelstein. The primary improvement over traditional random-primed method involves the use of random decamers instead of hexamers, ensuring more efficient annealing with DNA at 37°C. Klenow Fragment, exo-, included in the kit, is genetically engineered enzyme with no detectable exonuclease activity. The enzyme does not degrade the labeled probe during reaction, which results in a high labeling yield even with low amounts of template. You can uniformly label any length DNA fragments.

Biotin-labeled DNA is detected with the Biotin Chromogenic Detection Kit or conventional biotin-avidin or biotin-streptavidin detection systems.

Highlights

Non-radioactive labeling of DNA
Efficient priming of labeling reactions with random decamers
High yields with Klenow Fragment, exo-: no degradation of a labeled probe during reaction

Applications

• Generation of biotin-labeled DNA probes for a variety of non-radioactive hybridization experiments, including Southern and Northern blots, colony/plaque hybridizations, dot/slot blots, and in situ hybridizations.

Principle

Random decamers are annealed to a denatured template DNA molecule, and new strands are synthesized by the Klenow Fragment, exo- in the presence of biotin-dUTP. During this reaction, the biotinylated nucleotides are incorporated into the newly synthesized complementary DNA strand.

Related Products
Biotin DecaLabel DNA Labeling Kit

Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo Scientific™)

The Thermo Scientific Pierce RNA 3'-End Desthiobiotinylation Kit contains reagents for easy and efficient desthiobiotin labeling of RNA for use as probes or as antibody alternatives for enrichment of RNA binding proteins.

Features of the RNA 3'-End Desthiobiotinylation Kit:

Non-radioactive – a single biotin labels has similar detection sensitivity as radioactivity
Easy to use – RNA ligase and optimized reaction buffer are included
Economical – only a fraction of the cost purchasing synthetic biotinylated RNA probes
End-labeled – results in minimal disturbance of RNA secondary structure
Flexible – desthiobiotin label may be used for detection or as an affinity handle for streptavidin resin

The Pierce RNA 3'-End Desthiobiotinylation Kit is optimized for labeling the 3'-end of single-stranded RNA using T4 RNA ligase. Once labeled, the RNA can be used as a probe or target for gel-shift EMSA reactions, Northern blots and protein-RNA interaction experiments. The desthiobiotinylated RNA also can be used to enrich for RNA binding proteins (RBP) using streptavidin affinity resin, because the desthiobiotin tag binds to streptavidin in a manner which allows gentle elution of the ribonucleoprotein complex. The labeling kit is also included as a component of the Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit.

Includes:
Desthiobiotinylated cytidine bisphosphate, T4 RNA ligase and buffer, unlabeled RNA oligonucleotide for use as a positive control, a biotinylated RNA probe standard, RNase inhibitor, glycogen, and ligation enhancing reagents

Applications:
• Northern blotting
• EMSA
• Enrichment of RNA binding proteins (RBP)

This RNA labeling kit uses T4 RNA ligase to attach a single desthiobiotinylated cytidine bisphosphate to the 3-'end of single-stranded RNA. Each labeling reaction was designed for 50 pmol of RNA; however, reactions may be scaled (1 pmol to 1nmol have been tested), if necessary. The reaction uses a 20-fold excess of desthiobiotinylated nucleotide and requires incubation times from 30 minutes at 37°C (for less complex RNA) to overnight at 4 to 16°C (for longer or more complex RNA). Optimization of the labeling efficiency for complex RNA structures is achieved by altering the RNA:nucleotide ratio, increasing the incubation time, or by adding DMSO to relax the RNA structure. After organic extraction and precipitation with ethanol, the desthiobiotin-labeled RNA is ready for use in downstream applications.

Example experiments with different RNA molecules demonstrate the robustness of the ligation reaction. Synthetic RNA and in vitro transcribed RNA were evaluated, and ligations efficiencies were determined by semi-quantitative dot blot using a synthetic biotinylated RNA as the 100% labeled control (Table). Ligation efficiencies were all greater than 50%, with many greater than 70%. The kit control RNA had greater than 90% efficiency, and serves as an indicator of the overall reaction conditions and reagents. Further optimization was necessary for RNA having significant secondary structure, and refolding was required for in vitro transcribed RNA after ligation.

A pull-down assay involving the let-7 miRNA:Lin28 interaction demonstrates the experimental functionality of desthiobiotinylated-labeled RNA. The developmentally regulated Lin28 protein is a selective inhibitor of let-7 miRNA processing through binding to a loop region of let-7 pre-miRNA. To test this interaction, the loop portion of let-7 miRNA was end-labeled with desthiobiotinylated cytidine and attached to streptavidin magnetic beads. Beads were subsequently incubated with embryonal carcinoma cell lysate (NCCIT) in Protein-RNA binding buffer. Samples were eluted and assayed by Western blotting. The results indicate that the labeled let-7 miRNA specifically enriched Lin28 protein from NCCIT lysate, while an unrelated RNA did not.

This kit is also included in The Pierce Magnetic RNA-Protein Pull-Down Kit and utilizes the labeling kit in conjunction with optimized binding, wash, and elution buffers to enrich RNA binding proteins.

Ulysis™ Alexa Fluor™ 488 Nucleic Acid Labeling Kit (Invitrogen™)

ULYSIS® Nucleic Acid Labeling Kits provide a unique method to attach a fluorescent dye to nucleic acids. The labeling reagent in the kit reacts with the N7 of guanine to form a stable coordination complex, and the reaction is simple and fast—just heat denature DNA (5 minutes), add the label (react for 15 minutes), then purify.

ULYSIS® Nucleic Acid Labeling Specifications:
• Dye (Ex/Em): Alexa Fluor® 488 (492/520 nm)
• Labeling reaction is complete in as little as 15 minutes
• Available in several Alexa Fluor® dye colors


The Resulting Labeled Probe is useful for:
• Dot, northern, and Southern blots
• RNA and DNA in situ hybridization
• Multicolor fluorescence in situ hybridization (mFISH)
• Comparative genome hybridization (CGH)
• Microarray analysis

Reliable Labeling With the Universal Linkage System
We developed this series of ULYSIS® kits to enable rapid and simple coupling of our Alexa Fluor® dyes to purine bases in nucleic acid polymers. The method, the Universal Linkage System (ULS™), is based on the use of a platinum dye complex (owned by KREATECH Diagnostics) that forms a stable adduct with the N7 position of guanine and, to a lesser extent, adenine bases in DNA, RNA, PNA, and oligonucleotides. The result is a reliable nonenzymatic method for nucleic acid labeling.

Labeling is Fast and Easy
The labeling reaction typically takes only 15 minutes, and separation of the labeled nucleic acids from the unreacted ULS™ complex can be accomplished through the use of a simple spin-column procedure. DNA longer than ~1,000 base pairs requires a 10-minute DNase digestion before labeling, which both optimizes labeling and fragments the probe for efficient hybridization.

More Options for Nucleic Acid Labeling
To review various options for nucleic acid labeling, review Labeling Oligonucleotides and Nucleic Acids—Section 8.2 in the Molecular Probes® Handbook or view a list of our kits.

For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.

SuperScript™ One-Cycle cDNA Kit for use with Affymetrix™ One-Cycle Assays (Invitrogen™)

The SuperScript® One-Cycle cDNA Kit for use with Affymetrix® One-Cycle Assays conveniently contains all of the reagents required for double-stranded cDNA synthesis within the Affymetrix GeneChip® workflow. The kit includes Invitrogen’s proprietary RNase H reduced activity SuperScript® II Reverse Transcriptase and a qualified oligo (dt)-containing primer to perform reactions standard to Affymetrix’ protocol. Designed to consistently produce higher yields of full-length cDNA for Affymetrix One-Cycle assays using either total RNA or poly A+ selected RNA (mRNA).

BioPrime™ Total FFPE Genomic Labeling System (Invitrogen™)

The BioPrime® Total FFPE Genomic Labeling System is a complete genomic DNA labeling kit designed for use in array comparative genomic hybridization applications (aCGH) using formalin-fixed paraffin-embedded (FFPE) samples. Previous protocols have required large amounts of very high quality DNA, which is often difficult to obtain from FFPE material. With a random prime amplification (RPA) method, using labeling mixes specifically formulated for FFPE samples, BioPrime® Total FFPE Genomic Labeling System generates high yields of labeled DNA which is most representative of the starting sample.. Increased call accuracy is achieved with higher yields of labeled material on the array, giving you more accurate calls. The BioPrime® Total FFPE Genomic Labeling System offers users the following advantages:

- Improved call rate with higher signal to noise ratios from Alexa Fluor® 3 and 5 dyes
- Easy cleanup with PureLink™ purification eliminates the need for complicated volume-reduction steps
- Less channel bias due to enhanced reaction formulation for FFPE samples
- Lower requirements for input material using enzymatic RPA method


This complete, all-inclusive kit is optimized to work across a wider range of sample input material with no need for pre-amplification. Highly concentrated exo-Klenow fragment of DNA polymerase I in a component limited reaction allow for consistent, robust DNA yields of genomic FFPE DNA input. BioPrime® Total for Agilent® aCGH is supplied with Purelink™ purification which allows for low elution, eliminating the need for complicated volume-reduction steps. Purelink™ purification also reduces background due to dye bled found in other purification systems An optimized dye labeled nucleotide mix with new dNTP linkers, novel, application-specific Alexa Fluor® 3 and 5 dyes, and improved buffer chemistry reduce labeling variation and increase signal to noise on arrays. Excitation and emission spectra of Alexa Fluor® 3 and 5 dyes are suited for conventional two color scanners with no need to change settings. Complete kits are batch tested to assure quality in performance from lot to lot.

KinaseMax™ 5' End-Labeling Kit (Invitrogen™)

The Ambion® KinaseMax™ 5' End-Labeling Kit allows the efficient end-labeling of DNA or RNA to high-specific activity with T4 polynucleotide kinase and [gamma-32P] ATP, or quantitative phosphorylation of 5' ends using unlabeled ATP. Includes sufficient reagents for 30 reactions.

• Label oligonucleotide probes for nuclease protection assays and blot hybridizations
• Label primers for northerns and quantitative PCR
• Obtain up to 3-fold greater yields than with standard kinase buffers
• Reagents included for forward and dephosphorylation reactions

The kit includes a kinase reaction buffer that exceeds the performance of the standard forward reaction buffers recommended in "Molecular Cloning: A Laboratory Manual" (Sambrook et al., 1989) and "Current Protocols in Molecular Biology" (John Wiley & Sons, Inc. Ausubel, FM et al. editors, 1994). Crude 7000 Ci/mmol [gamma-32P]ATP is compatible with this kit. End-labeled probes are more stable than internally labeled probes and can routinely be used in assays for two to four weeks.

Rapid, Phenol-Free Phosphatase Removal Step
Inactivating the Calf Intestinal Phosphatase (CIP) after the dephosphorylation reaction is a time-consuming and cumbersome procedure that usually requires phenol extraction and ethanol precipitation typically resulting in sample loss. The KinaseMax™ Kit now includes a novel Phosphatase Removal Reagent for quick and complete removal of CIP. After the dephosphorylation step, the reagent is added to the reaction, incubated at room temperature for 2 minutes. The tube is then spun for a minute in the microfuge and the supernatant transferred to a new tube for the kinasing reaction.

Accessory Products:
The NucAway™ Spin Columns (SKU# AM10070) are available for rapid purification of end-labeled probes.

SuperScript™ Indirect cDNA Labeling Module, no purification columns (Invitrogen™)

The SuperScript® Indirect cDNA Labeling System is an array labeling kit based on proven methods of indirect cDNA labeling. The optimized system provides:

• SuperScript® III RT for generating high cDNA yields
• A proprietary nucleotide mixture to increase signal intensity
• A convenient kit format that saves valuable time

In this indirect labeling method, mRNA or total RNA is reverse transcribed using SuperScript® III RT, incorporating amino-modified dUTP and dATP into the synthesized cDNA. The template RNA is then degraded by base hydrolysis, and the reaction is neutralized with acid. The amino-modified cDNA is then purified to remove unincorporated nucleotides, primers, and buffers. In the second step, the modified cDNA is coupled with the active form of a fluorescent dye. The fluorescently labeled cDNA is purified with a S.N.A.P.™ spin column to remove any unreacted dye. The resulting fluorescently labeled cDNA is ready for hybridization to microarrays (Figure 1).

The SuperScript® Indirect cDNA Labeling System provides a complete system that includes both the core kit reagents (except dyes) and a purification module. The SuperScript® Indirect cDNA Labeling Module includes just the core kit reagents. Both provide the flexibility to use fluorescent nucleotides of your choice.

Pierce™ Biotin 3' End DNA Labeling Kit (Thermo Scientific™)

The Thermo Scientific Pierce Biotin 3' End DNA Labeling Kit is for tagging single-stranded DNA primers with biotin for use in non-radioactive electrophoretic mobility shift assays (EMSA) and other nucleic acid detection methods.

The DNA biotinylation procedure uses terminal deoxynucleotidyl transferase (TdT) to catalyze nontemplate-directed nucleotide incorporation onto the 3'-OH end of single-stranded DNA. TdT exhibits a substrate preference of single-stranded DNA, but it will label duplex DNA with 3' overhangs and blunt duplexes, albeit with a lower efficiency.

Features of the Biotin 3' End DNA Labeling Kit:

• Non-isotopic labeling eliminates the hassle of hazardous radioactive materials or difficult-to-dispose-of waste
• 1-3 biotinylated ribonucleotides onto the 3' end of DNA strands for less interference with hybridization or sequence-specific binding of proteins
• Biotin-labeled probes are stable for more than one year
• 30-minute labeling procedure is fast and efficient

The Biotin 3' End DNA Labeling Kit has been optimized to incorporate 1-3 biotinylated ribonucleotides (Biotin-11-UTP) onto the 3' end of DNA strands. This labeling strategy has the advantage of localizing the biotin to the 3' end of the probe where it will be less likely to interfere with hybridization or sequence-specific binding of proteins. Biotin-labeled DNA probes can be used to facilitate non-isotopic detection in a variety of applications including electrophoretic mobility shift assays (EMSA), Northern or Southern blots, colony hybridizations or in situ hybridizations.

BioPrime™ Array CGH Genomic Labeling Module (Invitrogen™)

The BioPrime® Plus Array CGH Genomic Labeling Systems are a high-performance labeling kits that enable reproducible labeling of genomic DNA samples for array-based Comparative Genomic Hybridization (CGH). Available in both indirect and direct labeling formats, the BioPrime® Plus Array CGH Genomic Labeling Systems provide a flexible solution to your genomic labeling needs. Using the
BioPrime® Plus Array CGH Genomic Labeling Systems, you can expect:
––High yields of fluorescently labeled genomic DNA
–signal-to-noise ratios
–detection of gene copy number variations (Figure 1)


These systems combine a highly concentrated exo-Klenow fragment of DNA polymerase I and random primers to effectively label genomic targets for sensitive genomic profiling experiments. Exo-Klenow is a mutant of the large fragment of the DNA polymerase I holoenzyme that has both 5´ - 3´ and 3´ - 5´ exonuclease activity removed. The lack of exonuclease activity makes this enzyme ideal for use in random priming protocols, enabling robust yields and efficient incorporation of Alexa Fluor® AHA fluorescent nucleotides. In the BioPrime® Plus Array CGH Genomic Labeling Systems, random octamers anneal to template DNA, providing priming sites for the exo- Klenow enzyme. Alexa Fluor® AHA modified nucleotides (direct labeling) or amino-allyl modified nucleotides (indirect labeling) are incorporated as the polymerase extends from the priming sites. The labeled samples are then purified to remove contaminants prior to denaturing and hybridization to a microarray (direct labeling systems) or coupled to the Alexa Fluor® NHS ester (indirect labeling systems) prior to hybridization (Figure 2).

SuperScript™ Plus Direct cDNA Labeling Module (no purification) with Alexa Fluor™-aha-dUTPs (Invitrogen™)

The SuperScript® Plus Direct cDNA Labeling System is a microarray labeling kit that combines the superior performance of SuperScript® III Reverse Transcriptase (RT) with the simplicity of direct labeling. In place of an enzyme spike-in, the streamlined protocol incorporates balanced sets of novel, array-optimized, nucleotides conjugated to Alexa Fluor® 555 and 647 dyes. This results in more reproducible array data with higher correlation coefficients than using standalone reagents. The SuperScript® Plus Direct cDNA Labeling System provides:

• Optimized, all-inclusive direct labeling with balanced aha-Alexa Fluor® nucleotides, enabling higher numbers of array positives
• Improved correlation coefficients for more accurate data (Figure 1)
• Low elution purification columns included for ease of use

In the SuperScript® Plus cDNA Labeling method, anchored oligo(dT) anneals to the mRNA template (Figure 2). SuperScript® III RT extends from the priming site, incorporating dUTP labeled with Alexa Fluor® dye directly to produce labeled cDNA. Template RNA is degraded by base hydrolysis. Free nucleotides and other contaminants are removed using low-elution volume purification columns before hybridization to the microarray.

BioPrime™ DNA Labeling System (Invitrogen™)

The BioPrime® DNA Labeling System is specifically designed for use in the preparation of biotinylated probes. Random primers (octamers) are annealed to the denatured DNA template and extended by Klenow fragment in the presence of biotin-14-dCTP to produce sensitive biotinylated-DNA probes for use in the nonradioactive detection of DNA and RNA. With the BioPrime® DNA Labeling System, considerable net DNA synthesis occurs resulting in a 10–40 fold amplification of the probe. This product is particularly well suited for situations in which the DNA is limited. Probes made with this product have been used for hybridization to Southern and northern blots, plaque and colony lifts, and for in situ hybridization to chromosome spreads. The BioPrime® DNA Labeling System:

• Requires less template DNA than nick translation
• Amplifies template to provide an increased amount of biotinylated probe
• Labels 50 to 500 ng of template DNA in one reaction