为核酸分析工作流程选择合适的电泳产品是确保您实验成功的关键。这些DNA/RNA分析常见问题的处理诀窍可帮助您改善电泳结果。
Tip 1: Choosing the right ladder
诀窍1：选择合适的分子量标准，对PCR产物或高通量凝胶的DNA条带进行分子量大小测定
这一类应用需要条带较少的DNA分子量标准。不妨试试Thermo Scientific FastRuler DNA分子量标准。该产品可以快速分离且迁移距离较短。FastRuler 分子量标准为五种平末端、色谱纯化的DNA片段混合物，在琼脂糖凝胶上运行8-14分钟后，可在较短的电泳距离(10–20 mm)轻松分离。
- Number of bands: Choose a ladder with the appropriate number of bands for the size range of your PCR product or high throughput gel
- Migration distance: Consider the migration distance of the ladder. For example, the FastRuler ladders have a short migration distance of 10–20 mm and are designed for fast separation after an 8–14 min run on an agarose gel.
- Purity: Look for ladders that are chromatography-purified to help ensure high purity and accurate results
- Resolution: Choose a ladder with good resolution to easily resolve DNA fragments
- Compatibility: Make sure the ladder is compatible with your gel system and run conditions, such as run time and voltage
- Quality control: Ensure that the ladder has been quality controlled and validated to help ensure reliable results
The right ladder can greatly impact the accuracy and efficiency of your experiment (Figure 1).
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Figure 1. High-throughput agarose gel electrophoresis using the FastRuler High Range DNA Ladder. DNA samples run alongside the appropriate DNA ladder, which shows clean separation of ladder markers.
图1. 高通量琼脂糖凝胶电泳（FastRuler High Range DNA分子量标准）.
Tip 2: Choosing the optimal agarose gel concentration
诀窍2：选择最佳的琼脂糖凝胶浓度
琼脂糖浓度对您的样品以及凝胶上的分子量标准的分离质量有较大影响。待分析DNA片段越长，所需的琼脂糖凝胶浓度越低（参见下方表1）。
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琼脂糖浓度及其对应的有效分离范围
| 琼脂糖浓度（%） | 有效分离的片段范围 |
|---|---|
| 0.5 | 2,000–50,000 bp |
| 0.6 | 1,000–20,000 bp |
| 0.7 | 800–12,000 bp |
| 0.8 | 800–10,000 bp |
| 0.9 | 600–10,000 bp |
| 1.0 | 400–8,000 bp |
| 1.2 | 300–7,000 bp |
| 1.5 | 200–3,000 bp |
| 2.0 | 100–2,000 bp |
| 3.0 | 25–1,000 bp |
| 4.0 | 10–500 bp |
| 5.0 | 10–300 bp |
Figure 2. Effect of agarose concentration on DNA resolution. (A) Poor band resolution resulting from incorrect agarose concentration. (B) Improved band resolution with correct agarose concentration.
图2. (A) 琼脂糖浓度影响DNA 分子量标准分离的分辨率。 (B) 根据待分析片段长度范围妥善选择琼脂糖浓度，可提升条带分辨度。
The two common running buffers used in DNA electrophoresis are TAE and TBE buffer solutions (Figure 3). Linear double-stranded DNA fragments migrate approximately 10% slower in TBE buffer when compared to TAE buffers.
诀窍3：选择最佳的电泳运行缓冲液
采用TAE还是TBE运行缓冲液？
TAE
- 分离较长的片段（通常用于>1 kb的片段），使用TAE缓冲液效果更好
- 与酶促反应物相容
- 推荐用于凝胶电泳制备
TBE
- 通常更适合分离较小的DNA片段
图3. 选择最佳的电泳缓冲液。在TBE缓冲液中，线性双链核酸片段的迁移速率大约下降10%。
The guidelines for choice of TAE and TBE buffers are:
Tris-Acetate-EDTA (TAE) Running Buffer
- Longer fragments are better resolved with TAE buffer (typically for fragments >1 kb)
- Compatible with enzymatic reactions
- Recommended for preparative gel electrophoresis
- Not suitable for longer runs
Tris-Borate-EDTA (TBE) Running Buffer
- Commonly used for better separation of small DNA fragments
- Not recommended for applications involving enzymatic steps
- Higher ionic strength makes it suitable for long runs
Tip 4: Choosing a proper sample loading dye/buffer
诀窍4：选择适当的样品上样染料/缓冲液
样品上样缓冲液有两个作用：
- 提供眼睛可见的染料，让您可以在凝胶上样时观察样品；电泳过程中还可形成染料前沿，让您可以看到凝胶电泳前进的距离。
- 其中含有高百分比的甘油，让样品比电泳缓冲液更重，从而沉到孔底，避免样品扩散到电泳缓冲液中。
选择上样缓冲液后，DNA样品和分子量标准务必采用同一种缓冲液。要避免因上样缓冲液中的示踪染料遮挡目的条带。例如，6X Orange DNA上样缓冲液含有Orange G（迁移性类似于50 bp DNA）和二甲苯蓝（迁移性类似于4,000 bp DNA）示踪染料。因此，尽管这种上样缓冲液通常非常适合于小片段电泳，但由于Orange G的干扰，约50 bp长度的条带可能不可见。
图4.择最适宜的凝胶电泳样品上样染料/缓冲液。
When selecting a dye, care must be taken to avoid masking bands of interest with the tracking dyes that are present in loading buffers. For example, 6X Orange DNA loading buffer contains the dyes Orange G and xylene cyanol. Orange G migrates like a 50 bp DNA fragment, and xylene cyanol migrates like 4,000 bp DNA fragment (Figure 4). Although this loading buffer is generally suitable for electrophoresis of small fragments, bands of ~50 bp fragments may not be visible due to masking by the Orange G.
Tip 5: Choosing the optimal sample quantity
诀窍5：选择最适宜的上样量
- 凝胶上的DNA上样过多，会影响样品的迁移。上样过多会降低片段电泳速率，导致测得的分子大小要超过实际的分子大小。
- 凝胶上的DNA上样太少，则会导致难以在凝胶上检测出靶标，尤其是较小条带会非常微弱。
如果采用溴化乙锭(EtBr)或SYBR Safe DNA凝胶染料对凝胶染色，请确保各个孔上样的DNA量至少达到20 ng/条带。SYBR Gold核酸凝胶染料要比EtBr或SYBR Safe DNA凝胶染料更灵敏，各个孔上样的DNA至少应达到1 ng/条带。
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- Too much DNA loaded on a gel can affect the migration of the sample; an overloaded fragment runs slower and therefore can seem to be larger in size than it really is (Figure 5)
- Too little DNA is hard to detect on a gel; the smaller bands appear faint (Figure 5)
Figure 5. Effect of DNA sample concentration on gel migration patterns. Increasing concentrations of DNA samples aliquoted into different wells on an agarose gel. Bands at 2,500 bp and 100 bp are shown.
图5. 样品浓度对迁移模式的影响。
Tip 6: Choosing the optimal gel size
诀窍6：选择理想的凝胶尺寸
- 小凝胶：常采用 8 x 10 cm凝胶（mini凝胶）。这种尺寸的凝胶成像非常方便。mini凝胶的琼脂糖溶液体积通常为30–50 mL。
- 大凝胶：较大的凝胶用于Southern和northern印迹等应用。这些凝胶的琼脂糖溶液体积应为大约250 mL。
Tip 7: Avoiding the “smiling” effect
When the DNA samples in the center lanes migrate faster than the peripheral lanes, the DNA bands form a crescent shape; this is called the “smiling” effect (Figure 6). The main causes of bands “smiling” on a gel are:
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- Uneven heating of the gel across different lanes, usually caused by high voltage. To avoid uneven heat distribution, you can run the gel slowly by reducing the voltage, so that temperature inconsistency is minimized.
- Uneven distribution of the electric field across the gel width. This can be addressed by checking the tank setup for loose contacts or other possible problems in the electrophoresis tank.
Figure 6. “Smiling” effect on a gel. DNA samples run via agarose gel electrophoresis may encounter the “smiling” effect where center samples run faster than outer-lane samples usually due to high voltage or loose contacts in the gel tank.
诀窍7：避免“微笑”效应
导致凝胶条带“微笑”效应的主要原因有：
- 不同泳道的凝胶不均匀受热。这通常是由于电压过高造成的。为避免出现这种情况，用户可以采用更低的速度运行凝胶（降低电压），从而最大限度减少温度不一致性。
- 电场在凝胶宽度范围内不均匀分布。检查电泳槽设置是否存在触头松动或其他潜在故障可以有效解决这一问题。
图6. 凝胶的“微笑”效应
Tip 8: Importance of gel immersion in the running buffer
A gel must be fully submerged in running buffer with 3–5 mm of buffer covering the gel’s surface. Insufficient amounts of running buffer can cause poor resolution, band distortion, or even melting of the gel (Figure 7). However, excess running buffer can decrease DNA mobility and cause band distortion.
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Figure 7. Volume of running buffer affects DNA mobility and band resolution. DNA samples run via agarose gel electrophoresis with insufficient amount of buffer. This contributes to the distortion of DNA bands and provides poor resolution.
诀窍8：凝胶浸入电泳缓冲液的重要性
凝胶必须完全浸入到电泳缓冲液中，保持液面没过凝胶表面至少3–5 mm。电泳缓冲液不足会导致分辨率下降、条带变形，甚至引起凝胶融解。而电泳缓冲液过多则会导致DNA迁移速度下降，同样会导致条带变形。
图7. 电泳缓冲液的体积会影响DNA迁移速度和条带分辨率。
仅供科研使用，不可用于诊断目的。