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查看更多产品信息 E-Gel™ Opener - FAQs (G530001)
11 个常见问题解答
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。
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.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
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.
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.