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          常見問題解答

          FAQ:?GelRed? and GelGreen? Nucleic Acid Gel Stains

          General GelRed?/GelGreen? FAQs

          Most issues that may arise from our GelRed? and GelGreen? nucleic acid stain products can be solved through the FAQ links found below. For more information about these products, please visit our DNA stains technology page.

          View the GelRed? Product Line

          View the GelGreen? Product Line

          Where can I find the expiration date or shelf life of a product?

          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. 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.

          My product was accidentally left out at room temperature or exposed to light. Is it ruined?

          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.

           

          What is the difference between GelRed? and GelGreen?? Which one should I choose?

          The main difference between GelRed and GelGreen is their fluorescence excitation and emission wavelengths. GelRed has red fluorescence, similar to ethidium bromide. GelGreen has green fluorescence, similar to SYBR? Green or SYBR? Safe. Both dyes are compatible with standard UV transilluminators. GelGreen is also compatible with blue light transilluminators, which allow users to avoid exposing themselves and their DNA samples to ultraviolet radiation.

          GelRed and GelGreen have higher sensitivity for double stranded nucleic acids compared to single stranded nucleic acids, but GelRed is more sensitive for staining single stranded nucleic acids than GelGreen. GelRed is about twice as sensitive for double stranded nucleic acids compared to single-stranded nucleic acids, and about five times more sensitive than GelGreen for staining single stranded nucleic acids.

          How do I use GelRed? and GelGreen??

          GelRed and GelGreen can be added to agarose during gel casting at a final concentration of 1X, or they can be used for post-electrophoresis gel staining at a final concentration of 3X in water. For detailed protocols for use, please download the GelRed? Product Information Sheet or GelGreen? Product Information Sheet.

          How many gels can I stain with a vial of GelRed? or GelGreen??

          GelRed and GelGreen can be added to agarose during gel casting at a final concentration of 1X, or used for post-electrophoresis gel staining at a final concentration of 3X in water. A 0.5 mL vial of GelRed or GelGreen is can be used to prepare 100 minigels (50 mL each) using the precast protocol, or for post-electrophoresis staining of 33 minigels in 50 mL staining solution per gel. Post-staining solution also can be re-used for staining two or more gels. For detailed protocols for use, please download the GelRed? Product Information Sheet or GelGreen? Product Information Sheet.

          Why am I seeing smeared or smiling DNA band(s) or discrepant DNA migration?

          Many customers use GelRed precast gels for convenience. However, because GelRed and GelGreen are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed precast gels. The following modifications may improve band resolution in precast gels.

          1. Reduce the amount of DNA loaded. Smearing and smiling is often caused by overloading of DNA. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA.
          2. Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
          3. Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.
          4. To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol.

          Why do I see weak fluorescence, decreased dye performance over time, or a film of dye remaining on the gel after post-staining?

          The dye may have precipitated out of solution.

          1. Heat GelRed solution to 45-50°C for two minutes and vortex to dissolve.
          2. Store dye at room temperature to avoid precipitation.

          Does post-staining require a de-staining step?

          No, but de-staining with water can be performed if background is high.

          Can GelRed? and GelGreen? be used to stain ssDNA or RNA?

          GelRed and GelGreen can be used to stain both ssDNA and RNA, but GelRed is about 5 times more sensitive for single-stranded nucleic acids than GelGreen. Titration assays using a fluorescence microplate reader showed that the fluorescence signal of GelRed bound to ssDNA and RNA is about half that of GelRed bound to dsDNA.

          What instruments can be used to detect GelRed? and GelGreen??

          GelRed is compatible with a standard UV transilluminator (302 or 312 nm).
          GelGreen has sufficient absorption between 250-300 nm and a strong absorption peak at around 500 nm. GelGreen is compatible with a 254 nm UV transilluminator or a gel reader with visible light excitation such as a Dark Reader or a 488 nm laser gel scanner.

          What emission filters are suitable for use with GelRed? and GelGreen??

          Use the ethidium bromide filter for GelRed; use a SYBR Green or yellow filter for GelGreen. Alternatively, a long-pass yellow filter can be used with both GelRed and GelGreen. Please review the emission spectra for GelRed and GelGreen for more specific wavelengths.

          Can I make GelRed/GelGreen gels ahead of time and store them for later use?

          You can store precast GelRed gels for up to a week, and GelGreen gels for up to a month. We recommend storing gels at room temperature in the dark. We no longer recommend storing gels at 4°C, because this can lead to dye precipitation and poor performance.

          What is the stability of GelRed?/GelGreen? in molten agarose?

          GelRed is more stable than GelGreen. We do not recommend storing GelRed in molten agarose for more than a few days.

          Can I reuse a GelRed?/GelGreen? precast gel after running samples?

          No. We do not recommend that GelRed/GelGreen gels be reused after electrophoresis because the staining intensity can be decreased with sequential electrophoresis.

          Can I re-melt a GelRed?/GelGreen? gel and cast again?

          Yes, but it may be necessary to add some more dye to the re-melted gel for the best signal.

          How should I dispose of GelRed? and GelGreen??

          GelRed and GelGreen passed the EPA regulated Title 22 test. Some facilities have approved the disposal of GelRed and GelGreen directly down the drain. However, because regulations vary, please contact your safety office for local disposal guidelines. Please review the GelRed/GelGreen safety report for more detailed information.

          Are GelRed? and GelGreen? compatible with downstream applications such as cloning, ligation and sequencing?

          Yes. We recommend Qiagen or Zymoclean gel extraction kits or phenol-chloroform extraction to remove the dye from DNA. Some users have reported performing PCR on DNA in the presence of GelRed with no purification step, for example by incubating GelRed-stained gel slices in TE buffer to extract DNA by passive diffusion for use in PCR, or by using a few microliters of molten agarose from GelRed-stained gel slices containing DNA for PCR.

          How safe is GelRed?/GelGreen??

          In AMES and related tests, GelRed and GelGreen were shown to be much safer alternatives to EtBr and SYBR dyes. Nevertheless, please exercise safe laboratory practices when using these reagents.

          Where can I find more information about the safety of GelRed?/GelGreen??

          Please visit our website www.biotium.com to download a comprehensive safety report.

          What is the lower detection limit of GelRed?/GelGreen??

          GelRed and GelGreen are ultra-sensitive dyes. Some users have reported being able to detect bands containing less than 0.1 ng DNA. However, the sensitivity of the staining will depend on the instrument capability and exposure settings.

          What is the binding mechanism of GelRed?/GelGreen??

          GelRed has been shown to bind DNA exclusively by intercalation (http://link.springer.com/article/10.1007/s00249-014-0995-4). GelGreen most likely binds by a combination of intercalation and electrostatic interaction.

          In which direction does GelRed?/GelGreen? migrate?

          GelRed and GelGreen do not migrate through the gel as easily as ethidium bromide. It is not necessary to add additional dye to the running buffer, and the gel will be stained more homogenously than a gel stained with ethidium bromide.

          Do GelRed? and GelGreen? need to be used in the dark?

          GelRed and GelGreen are stable dyes. You can use the dyes in room light. However, we recommend storage of the dyes in the dark between uses. We have had a customer report using GelRed with success after accidentally leaving it in ambient light for one month.

          How much GelRed?/GelGreen? do I need to use?

          Dilute the 10,000X stock 10,000-fold for 1X precast gels (for example, 5 uL for a 50 mL gel), or 3,333-fold for a 3X post staining solution (15 uL for a 50 mL solution).

          Can GelRed?/GelGreen? post-staining solution be reused?

          Yes. However, if the sensitivity decreases, use a fresh solution of the dyes.

          Is GelRed? compatible with alkaline gel running buffer (30mM NaOH, 1mM EDTA)?

          Yes.

          What purification protocols are recommended to remove GelRed?/GelGreen? after staining?

          Customers report good results using ZymoClean Gel DNA Recovery Kit from Zymo Research, GenElute Agarose Spin Column from Sigma, PureLink Quick Gel Extraction kit from Life Technologies, Illustra GFX PCR DNA and Gel Band Purification kit from GE Healthcare, High Pure PCR Product Purification Kit from Roche Applied Sciences and GenJET gel extraction kit from Thermo Scientific.

          Why do you offer GelRed? and GelGreen? in DMSO or water?

          The water formulation is a newer and improved product compared to the stock in DMSO. We recommend using dyes in water to avoid the potential hazards of handling DMSO, which can be absorbed through the skin. We continue to offer dyes in DMSO because some users do not wish to alter their established laboratory protocols. Based on internal testing, both formulations perform similarly.


          Application-Specific GelRed?/GelGreen? FAQs

          Where can I find the expiration date or shelf life of a product?

          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. 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.

          Can GelRed? and GelGreen? be used to stain ssDNA or RNA?

          GelRed and GelGreen can be used to stain both ssDNA and RNA, but GelRed is about 5 times more sensitive for single-stranded nucleic acids than GelGreen. Titration assays using a fluorescence microplate reader showed that the fluorescence signal of GelRed bound to ssDNA and RNA is about half that of GelRed bound to dsDNA.p>

          Are GelRed? and GelGreen? compatible with downstream applications such as cloning, ligation and sequencing?

          Yes. We recommend Qiagen or Zymoclean gel extraction kits or phenol-chloroform extraction to remove the dye from DNA. Some users have reported performing PCR on DNA in the presence of GelRed with no purification step, for example by incubating GelRed-stained gel slices in TE buffer to extract DNA by passive diffusion for use in PCR, or by using a few microliters of molten agarose from GelRed-stained gel slices containing DNA for PCR.

          Can GelRed?/GelGreen? be used for Southern or Northern blotting?

          GelRed has been validated for Southern blotting. We recommend using the post-staining protocol for blotting applications.

          Can GelRed? be used in formaldehyde based RNA gels?

          Yes, customers have reported using GelRed in glyoxal and formaldehyde agarose gels for precast staining of RNA.

          Can GelRed? be used for polyacrylamide, DGGE, EMSA or PFGE (pulse-field) gels?

          Yes. Please use the post-staining procedure for DGGE and EMSA gels. For PFGE gels, the pre-cast or post-staining protocol may be used.

          Can GelRed?/GelGreen?be used for Comet Assay?

          Yes.

          Can GelRed? be used for cesium chloride gradient purification of DNA?

          Customers have reported using GelRed in cesium gradients. To extract GelRed from DNA after cesium banding, we recommend adding SDS to a final concentration of 0.1% before butanol extraction. Alternatively, chloroform can be used instead of butanol for extraction.

          Can GelRed? be used to detect ssDNA in a precast gel?

          Can GelRed? be used in Loop-mediated Isothermal Amplification Assay?

          Can GelGreen? be used in Capillary Gel Electrophoresis-Laser induced Fluorescence of double-stranded DNA fragments?


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