GeneGelRedTM Nucleic Acid Gel Stain FAQs
1. Why do you offer GeneGelRed 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.
2. What instruments can be used to detect GeneGelRed? GeneGelRed is compatible with a standard UV transilluminator (302 or 312 nm).
3. What emission filters are suitable for use with GeneGelRed? Use the ethidium bromide filter for GeneGelRed; use a SYBR® Green or yellow filter for GelGreen®. Alternatively, a long-pass yellow filter can be used with both GeneGelRed. Please review the emission spectra for GeneGelRed for more specific wavelengths.
4. How safe is GeneGelRed? In AMES and related tests, GeneGelRed® was shown to be much safer alternatives to EtBr and SYBR dyes. Nevertheless, please exercise safe laboratory practices when using these reagents.
5. How should I dispose of GeneGelRed? GeneGelRed passed the EPA regulated Title 22 test. Some facilities have approved the disposal of GeneGelRed directly down the drain. However, because regulations vary, please contact your safety office for local disposal guidelines. Please review the GeneGelRed safety report for more detailed information.
6. How do I use GeneGelRed? GeneGelRed® 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.
7. Why am I seeing smeared or smiling DNA band(s) or discrepant DNA migration? Many customers use GeneGelRed precast gels for convenience. However, because GeneGelRed is 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 GeneGelRed precast gels.
Tip #1: Load less DNASmearing and smiling in GeneGelRed precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. 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. This usually solves band migration problems.
Tip #2: Try the post-staining protocolTo 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. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused.
8. Why do I see weak fluorescence, decreased dye performance over time, or a film of dye remaining on the gel after post-staining?There are a few possibilities: The dye may have precipitated out of solution. Heat the GeneGelRed solution to 45-50°C for two minutes and vortex to dissolve. Store dye at room temperature to avoid precipitation. If you are seeing high background staining of the gel, the agarose that you are using may be of low quality. Contaminants in the agarose may bind to the dye, resulting in increased background.
9. Can I make GeneGelRed gels ahead of time and store them for later use? Yes. You can store precast GeneGelRed gels for up to a week. We recommend storing gels at room temperature in the dark. Do not store the gels at 4°C, because this will lead to dye precipitation and poor performance.
10. How much GeneGelRed do I need to use? How many gels can I run with a vial of GeneGelRed? A 0.5 mL vial of GeneGelRed 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 2 or more gels.
11. What is the stability of GeneGelRed in molten agarose? We do not recommend storing GeneGelRed in molten agarose for more than a few days.
12. Do GeneGelRed need to be used in the dark? GeneGelRed is a stable dye. You can use it in room light. However, we recommend storage of the dye in the dark between uses.
13. Can GeneGelRed post-staining solution be reused? Yes. However, if the sensitivity decreases, use a fresh solution of the dye.
14. Does post-staining require a de-staining step? No, but de-staining with water can be performed if background is high.
15. What DNA ladders and loading buffers can I use with GeneGelRed? We have tested a variety of DNA ladders from different suppliers with GeneGelRed with good results. Similarly, any common DNA loading buffers can be used. Band migration problems or smearing or smiling DNA bands are most commonly caused by overloading of ladder. If you are experiencing band migration problems with your ladder, please try reducing the amount of ladder you load. We recommend loading 50-200 ng ladder per lane for GeneGelRed precast gels.
16. Can I reuse a GeneGelRed precast gel after running samples? No. We do not recommend reusing GeneGelRed gels after electrophoresis because the staining intensity can be decreased with sequential electrophoresis.
17. Can I re-melt a GeneGelRed gel and cast again? Yes, but it may be necessary to add some more dye to the re-melted gel for the best signal.
18. Is GeneGelRed 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.
19. Can I use GeneGelRed to stain ssDNA or RNA? GeneGelRed can be used to stain both ssDNA and RNA. Titration assays using a fluorescence microplate reader showed that the fluorescence signal of GeneGelRed bound to ssDNA and RNA is about half that of GeneGelRed bound to dsDNA.
20. What is the lower detection limit of GeneGelRed? GeneGelRed is 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.
21. What is the binding mechanism of GeneGelRed? GeneGelRed binds DNA exclusively by intercalation (http://link.springer.com/article/10.1007/s00249-014-0995-4).
22. In which direction does GeneGelRed migrate? GeneGelRed 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.
23. Can GeneGelRed be used for Southern or Northern blotting? GeneGelRed has been validated for Southern blotting. We recommend using the post-staining protocol for blotting applications.
24. Can GeneGelRed be used in formaldehyde based RNA gels? Yes, customers have reported using GeneGelRed in glyoxal and formaldehyde agarose gels for precast staining of RNA.
25. Can I use GeneGelRed in 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.
26. Can GeneGelRed be used for Comet Assay? Yes, GeneGelRed can be used for Comet assay.
27. Is GeneGelRed compatible with alkaline gel running buffer (30mM NaOH, 1mM EDTA)? Yes, GeneGelRed is compatible with alkaline running buffer.
28. Can GeneGelRed be used for cesium chloride gradient purification of DNA? Customers have reported using GeneGelRed in cesium gradients. To extract GeneGelRed 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.
29. What purification protocols are recommended to remove GeneGelRed 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.
30. Can I use GeneGelRed to detect ssDNA in a precast gel? Yes, see Ana. Biochem. doi: http://dx.doi.org/10.1016/j.ab.2012.09.003.
31. Can GeneGelRed be used in Loop-mediated Isothermal Amplification Assay? Yes, see Virol J. 2012, 9, 110.
32. Can GeneGelRed be used to stain DNA in acrylamide gels? Yes, use the post-staining protocol for polyacrylamide gels. For polyacrylamide gels containing 3.5-10% acrylamide, typical staining time is 30 minutes to 1 hour with gels of higher acrylamide content requiring longer staining time.,
33. 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.