Novex™ TBE-Urea Gels, 6%

这里有一些对您实验的建议：

•RNA样本可能被RNA酶降解了。遵循标准注意事项以避免污染。
•确保在上样前对样本在70°C加热3分钟。如果没有这样做，则寡核苷酸不会被完全变性，导致出现拖尾。
•确保在上样前彻底将尿素冲洗出上样孔。尿素会持续从凝胶渗入上样孔内。尿素很浓而且会使样本形成球状。
•上样体积超过10 µL可能引起拖尾。这一体积比琼脂糖凝胶所用的上样体积小。
•如果没有使用超纯水（18毫欧姆），则可能出现拖尾条带和高背景。
•检查电泳缓冲液和上样缓冲液是否正确制备和稀释。
•确保使溴酚蓝染料迁移到达边缘。过早停止电泳可能导致电泳效果较差。

该系统可以使用多种上样缓冲液配方；但是，我们发现根据上样缓冲液的不同，电泳条带的形态会有很大的不同。在评估了尿素、福尔马林以及各种缓冲液系统之后，我们发现使用尿素、Ficoll和TBE缓冲溶液能获得最清晰、最整齐的条带。使用含福尔马林的上样缓冲液将得到模糊、不清晰的条带。

可以，对于EB染色，将凝胶在超纯水配制的2 µg/mL EB溶液中浸泡20分钟。接下来使用超纯水进行3次洗涤脱色，每次10分钟。在紫外光下观察条带。

Here are some suggestions for your experiments:

- The RNA samples may have been degraded by RNases. Use standard precautions to prevent contamination.
- Make sure samples are heated for 3 minutes at 70°C just prior to loading. If this is not done, the oligonucleotides will not be fully denatured, which may result in a smeared background.
- Be sure to vigorously flush urea out of sample wells just prior to loading the sample. Urea will continually seep from the gel into the well. Urea is very dense and will force the sample into a ball.
- A sample volume over 10 µL may result in smearing. This volume is less than may be used with agarose gels.
- If ultrapure water (18 milliohms) is not used, smeared bands and high background may result.
- Check to make sure the running and sample buffers have been prepared and diluted correctly.
- Make sure to run the bromophenol blue until it reaches the slot. Stopping the run short can result in less than optimal results.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

There are many sample buffer formulations used; however, we have found a distinct difference in the band appearance depending on the sample buffer composition. After evaluating urea, formamide, and various buffer systems, we found that the sharpest, flattest bands were obtained with a urea, Ficoll, and TBE buffer solution. Sample buffers made with formamide provided fuzzy, indistinct bands.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.