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查看更多产品信息 E-Gel™ Agarose Gels with SYBR Safe™ DNA Gel Stain Starter Kit, 2% - FAQs (G620602)
27 个常见问题解答
与溴化乙锭相似，SYBR Safe DNA凝胶染料与DNA的迁移方向相反。由于只有凝胶的最底部染料浓度会在电泳过程中降低，所以使用SYBR Safe DNA凝胶染料制备的凝胶没有实际影响。在电泳结束后用SYBR Safe DNA凝胶染料染胶，可部分抵消这种作用。染料溶液不应该加入到电泳缓冲液中，因为这可能会导致染料在电极上分解，并将有毒的挥发性化合物释放到空气中。
请检查 E-Gel胶盒和铜电极。你可以换一个新的胶盒看看是否接触不良。过冷的胶盒或不恰当的操作条件也可能造成这个问题。胶盒应在室温使用，避免将其保存在4°C。
首先，检查你的琼脂糖凝胶的浓度。较高浓度的凝胶能帮助你分离较小分子量的分子，而较低百分比的凝胶能帮你分离较大分子量的分子。
每个泳道或者每个条带推荐的DNA上样量为20–100 ng。对于大部分E-Gel琼脂糖凝胶来说每个条带DNA上样量最多为500ng，而对于使用SYBR Safe染料的E-Gel凝胶，每条带的DNA的上样量最多为700ng。
完整RNA的28S:18S条带亮度比例应为2:1。你可能会在0.5 kb至9kb区间看到模糊的RNA拖尾条带，这表明样本中含有mRNA。请点击此处(https://www.thermofisher.com/cn/zh/home/references/ambion-tech-support/rna-isolation/tech-notes/assessing-rna-quality.html)查看图片或了解更多评估RNA质量的信息。
对于非变性RNA电泳，我们推荐使用 E-Gel 预制胶电泳系统。请注意E-Gel 琼脂糖凝胶并不保证完全不含RNA酶。但是，我们许多用户经常使用E-Gels琼脂糖凝胶成功的完成了RNA分析。如果使用E-Gel琼脂糖凝胶进行RNA电泳，可以使用任何适用于非变性DNA电泳的上样缓冲液。
对于变性RNA电泳，可以从以下变性试剂中进行选择，包括甲醛、乙二醛、甲酰胺、甲基汞。变性条件会破坏氢键，因此RNA在电泳时没有二级结构，作为单链分子而迁移。
对变性RNA电泳，我们的E-Gel EX琼脂糖凝胶可以使用。唯一与E-Gel EX系统兼容的变性剂是甲酰胺，50-95%。使用其他变性剂会导致条带分离较差，带型也不理想。请注意，我们不建议将在RNA上样缓冲液中制备的样品与在水中制备的样品在同一凝胶上电泳。RNA上样缓冲液配方及变性电泳条件如下：
RNA上样缓冲液：
去离子化甲酰胺：200 μL
10X MOPS-EDTA-乙酸钠缓冲液（0.4 M MOPS, pH 7.0，0.1 M乙酸钠，10 mM EDTA)：40 μL
去离子化甲醛：76 μL
水：14 μL < br / > < br / >
变性电泳条件：< br / >
1. 将15 μL RNA上样缓冲液与1-5 μL RNA (1-5 μg)混合。< br / >
2. 在65摄氏度下加热10分钟使RNA变性。< br / >
3. 加热后立即将样品放在冰上。< br / >
4. 将全部样品加载到E-Gel EX琼脂糖凝胶上。
5. 电泳30分钟。
对于不用甲醛作为变性剂的RNA变性电泳，我们推荐使用 NorthernMax-Gly 试剂盒（货号AM1946）。使用此试剂盒时，RNA样本在乙二醛／DMSO上样缓冲夜中变性，然后在含有乙二醛的琼脂糖凝胶中进行电泳。
琼脂糖因为无毒、易用且分离范围大而被广泛使用。我们提供预制E-Gel琼脂糖凝胶，也提供试剂用于配制琼脂糖凝胶。聚丙烯酰胺凝胶通常用于对10–3,000bp大小的DNA分子进行高分辨率分析。我们提供预制Invitrogen TBE聚丙烯酰胺凝胶和UltraPure试剂。
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.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
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.
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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).
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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).
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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.
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Yes, SYBR Safe stain is easily removed from nucleic acids by ethanol precipitation or by the ethanol wash steps used for purification spin columns.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.