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View additional product information for E-Gel™ Agarose Gels with SYBR Safe™ DNA Gel Stain Starter Kit, 1.2% - FAQs (G620601)
20 product FAQs found
Similarly to ethidium bromide, SYBR Safe DNA Gel Stain runs in the opposite direction of the migrating DNA. This has no practical effect on the use of gels cast with SYBR Safe DNA Gel Stain, as only the very bottom of the gel will have a lower concentration of stain. This effect can be partially counteracted by staining the gel with SYBR Safe DNA Gel Stain after electrophoresis. Solutions of dye should not be added to the running buffer as this can cause breakdown of the dye at the electrodes and release toxic volatile compounds into the air.
Check the E-Gel cassette and copper contacts. You can try replacing the gel cassette with a fresh gel cassette to see whether this fixes the problem. A cold cassette or improper operating conditions can also lead to this failure. Cassettes should be at room temperature for use; avoid storing at 4°C.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
First check the percentage of your agarose gel. A higher percentage will help you to resolve smaller molecular weights while a lower percentage will help you to resolve larger molecular weights.
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To determine the amount of DNA to load per well for your specific E-Gel agarose gel, please refer to the table on page 14 of the E-Gel Power Snap Electrophoresis System user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017050_egel_powersnapsystem_UG.pdf).
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Intact RNA should have a 2:1 ratio of 28S:18S bands. You may see a smear of RNA that extends from <9 kb to 0.5 kb, indicating the presence of mRNA in the sample. To see an image or to read more about RNA assessment, visit this website (https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/agarose-gel-electrophoresis-of-rna.html).
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
For nondenaturing RNA electrophoresis, we recommend using our E-Gel Precast Agarose Gels. Please note that E-Gel Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.
For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.
For denaturing RNA electrophoresis, our E-Gel EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:
RNA Loading Buffer:
Deionized formamide: 200 µL
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10 mM EDTA): 40 µL
Deionized formaldehyde: 76 µL
Water: 14 µL
Denaturing Electrophoresis Conditions:
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel EX agarose gel.
5. Electrophorese for 30 minutes.
For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Agarose is commonly used as it is nontoxic, easy to use, and offers a broad range of separation. We offer precast E-Gel Agarose Gels or reagents to pour your own agarose gels. Polyacrylamide gels are typically used for high resolution of DNA molecules that range in size from 10-3,000 bp. We offer precast Invitrogen TBE polyacrylamide gels and UltraPure reagents.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Yes, SYBR Safe stain is easily removed from nucleic acids by ethanol precipitation or by the ethanol wash steps used for purification spin columns.
Several E-Gel products are available with SYBR Safe DNA gel stain. These gels can be used in the same manner as their ethidium bromide counterparts, with the additional safety and application benefits of SYBR Safe. To learn more about these products, search "E-Gel Precast Agarose Gels" from the Thermo Fisher Scientific website home page.
We have found a distinct advantage to using SYBR Safe stain rather than ethidium bromide when purifying DNA from gels for downstream use. SYBR Safe stain is compatible with blue light imaging systems as well as UV. Using blue light to visualize the DNA allows you to purify a band with virtually no UV-induced nicking or crosslinking. This can dramatically increase cloning efficiency. Data from one such experiment showing higher cloning efficiency with PCR products visualized with SYBR Safe and blue light vs. ethidium bromide and UV light can be seen on the information page for Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600) and on the SYBR Safe home page.
We recommend that SYBR Safe stain be protected from light during storage and gel staining. However, it is sufficiently stable to withstand UV illumination for >30 minutes; realistically, hours of constant UV or bright room light exposure are required to cause any significant loss of signal.
SYBR Safe stain yields the same sensitivity as ethidium bromide - roughly 500 pg/band in a minigel for fragments larger than 200 bp viewed on a 300 nm transilluminator.
Many whitening agents used in clothing, some synthetic fibers, as well as some fungi and bacteria, fluoresce at the same wavelength as SYBR Safe stain. These contaminants within or on the surface of the gel may produce this “speckling”. This can be avoided by being careful with preparation of the gel (i.e. try to keep the gel dust free). Alternatively, to obtain a publication quality image you may be able to preferentially photobleach some contaminants by leaving the gel on the UV box for 15-30 minutes, where the speckles will disappear before the SYBR stain photobleaches.
SYBR Safe DNA gel stain has two main excitation peaks: in the UV region at 280 nm, and in the visible region at 502 nm. Thus, 254 nm or 300 nm UV excitation will work, as will 488 nm lasers, 470 nm LEDs, and broad blue excitation (such as the Safe Imager 2.0 Blue-Light Transilluminator, Cat. No. G6600). Maximal excitation occurs at 502 nm; the Safe Imager 2.0 Blue-Light Transilluminator is therefore the best choice for excitation of SYBR Safe DNA gel stain. The full excitation and emission spectra for SYBR Safe DNA gel stain are provided online and can also be found in the protocol.
Some ethidium bromide filters allow the transmission of all light above 500 nm. These filters (which are often yellow in color) and their associated camera settings can be used with SYBR Safe DNA gel stain, usually with only minor adjustments to the exposure or gain. Other ethidium bromide filters (often red in color) only transmit light around or above 600 nm; these filters and their associated camera settings are not suitable for use with SYBR Safe DNA gel stain.
Stained gels can be photographed using Polaroid 667 black-and-white print film and SYBR Safe photographic filter (Cat. No. S37100). Invitrogen SYPRO photographic filter (Cat. No. S6656) or a Kodak Wratten #9 filter also work well. Table 5 in the SYBR Safe product manual lists a filter selection guide for different instruments.
Please see the SYBR Safe home page for a list of recommended filters and settings for several different gel documentation instruments. You can find it by searching "SYBR Safe DNA Gel Stain" from the Thermo Fisher Scientific website home page.
If your system is not listed, please contact the instrument manufacturer for a recommendation. Note that the excitation and emission spectra of SYBR Safe gel stain are very similar to those of SYBR Green I, SYBR Green II, and SYBR Gold gel stains, as well as fluorescein (FITC). Therefore, filters appropriate for these dyes can also be used.
The SYBR Safe photographic filter (Cat. No. S37100) is a Wratten #9 gelatin filter. This filter is a 75 mm x 75 mm sheet of plastic that should be mounted in front of the lens of the camera. With a Polaroid camera and B&W film (#667), the filter may be taped inside the hood or mounted in a cassette and snapped in place inside the hood (the opening in which the camera lens is mounted upon).
For other camera systems, this sheet may be mounted in a cassette or filter-housing and placed in front of the camera lens. An alternative is to use thread-on glass filters of the same rating available from most camera supply vendors. Please note - not all camera systems require use of the SYBR Safe filter. See the SYBR Safe home page for a list of recommended filters and settings for several different instruments - you can find the page by searching "SYBR Safe DNA Gel Stain" from theThermo Fisher Scientific website home page.
DNA bands stained with SYBR Safe stain may be visible by eye on a 300 nm transilluminator if there is a sufficient amount of DNA per band. However, optimal detection is obtained by photographing the gel using a Wratten #9 emission filter (or other filter with a similar rating) with your CCD or film camera. With UV transilluminators (light boxes), UV bulbs may also emit some infrared (IR) wavelengths; if your camera lens is not specially coated to block IR, an IR-blocking filter is needed to prevent the appearance of the UV bulbs under your gel.
Migration speed through gels is correlated to the compactness of the DNA molecule. Circular DNA can exist as a relaxed circle (if one of the strands is nicked), and this form migrates very slowly. If the DNA circle is supercoiled, it adopts a more compact form and will migrate much more quickly. If both strands of the DNA circle are cleaved and the molecule is linear, that form will also migrate more quickly than the relaxed circle, but not as quickly as the supercoiled circle.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.