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DNA Quantitation Kits

AccuGreen™ kits are designed for use with the Qubit® fluorometer. All of the other kits are designed for use with 96-well fluorescence plate readers. Our quantitation kits vary in the concentration ranges of dsDNA that they are able to detect. Some of the kits also have different fluorescence excitation/emission. Please see the AccuBlue, AccuClear and AccuGreen Technology Highlights to choose the kit that best fits your application.

The AccuClear™ and AccuBlue™ DNA Quantitation kits are designed for use with fluorescence 96-well plate readers. AccuClear™, AccuBlue™ NextGen and AccuBlue™ High Sensitivity kits require an instrument equipped to read green fluorescence emission (similar to FITC). AccuBlue™ Broad Range requires an instrument equipped to read blue fluorescence emission (Ex/Em 350/460 nm).

These assays also can be used with fluorometers such as the Qubit® (Thermo Scientific) and QuantiFluor™-P (Promega). However, due to different linear ranges of the assays, not all of these assays are compatible with the pre-programmed DNA quantitation programs on these instruments. For users who own a Qubit® fluorometer, we recommend using our AccuGreen™ kit, which is designed for use on that instrument.

The AccuGreen™ High Sensitivity DNA Quantitation kit is designed for use on the Qubit® fluorometer. It can be used in the preprogrammed Qubit® dsDNA High Sensitivity assay, and is a direct replacement for the Qubit® dsDNA HS Assay kit.

For detection of picogram levels of DNA on a microplate reader, we recommend our AccuBlue™ NextGen dsDNA Quantitation Kit (detection range 2.5 pg-3 ng dsDNA) which has higher accuracy and sensitivity than PicoGreen® in a 96-well assay format.

Also see our AccuClear™ Ultra High Sensitivity dsDNA Quantation Kit, which is a great all-purpose choice: it has high sensitivity with a broad linear detection range (30 pg-250 ng dsDNA).

If you own a Qubit® fluorometer, we recommend our AccuGreen™ High Sensitivity dsDNA Quantitation kit, which is designed for use with the Qubit® fluorometer. It is a direct replacement for the Qubit® dsDNA HS Assay kit.

Note that the lower limits of kit sensitivity may depend on the detection instrument used.

A Quantitation Kit contains the quantitation solution components and pre-diluted calf thymus DNA standards. The Quantitation Solution contains the quantitation solution components, and does not include DNA standards.

No. It is recommended to use a DNA standard that most closely resembles your samples. For example, for bacterial DNA quantitation, you may prefer to use lambda DNA as a standard.

Yes, for some of the kits. You can purchase a set of AccuBlue™ High Sensitivity standards (cat. no. 31006C) or AccuBlue™ Broad Range standards (cat. no. 31007C) separately.

The dyes are selective for dsDNA; however there is some binding to RNA and ssDNA. Therefore, for the most accurate quantitation, it is recommended to use as clean a dsDNA preparation as possible. Please see the product information sheet for detailed information on RNA and ssDNA binding.

To minimize cost, the trial size kits come with a single DNA standard solution and instructions for preparing a set of standards by dilution. Most of the full size kits are provided with prepared standard sets. The exception is the AccuBlue™ NextGen kit, which is supplied with a single standard of 10 ng/uL, with instructions on diluting it to the lower concentrations used in the assay. We found that lower concentrations of DNA were not stable enough for long-term storage.

The AccuBlue™ High Sensitivity dye is membrane impermeable to live cells, while PicoGreen® has been shown to readily enter cells. We have not performed such testing with the dyes from our other DNA quantitation kits.

It is highly recommended to minimize cross-over of signals between wells. Depending on your instrument, you can use all black plates or plates with black-walls and clear bottoms.

Some of our DNA quantitation kits contain an enhancer in addition to the DNA quantitation dye. These kits have been optimized with the enhancer to maintain sensitivity and linearity. Omitting the enhancer may affect your results.

Yes. It is recommended to use as clean a dsDNA preparation as possible, but the kits can tolerate some common contaminants. Each kit varies in how it affected by various contaminants. Please see the product information sheets for each kit more information.

The assays can be scaled down for different well sizes. Scale the volumes of all reagents and DNA proportionally. The ratios of DNA, dye, and enhancer (if applicable) should remain the same as in the standard 96-well protocol.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

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