Choosing the right electrophoresis products for your nucleic acid analysis workflow is critical to the success of the experiment. Improve your electrophoresis results with these tips on commonly experienced issues in DNA/RNA analysis.
Tip 1: Choosing the right ladder for sizing PCR products or high-throughput gels
These applications require DNA ladders that have fewer bands. Try Thermo Scientific FastRuler DNA ladders, which are designed for fast separation and short migration distances. FastRuler ladders are a mixture of five blunt-ended, chromatography-purified DNA fragments that can be easily resolved over a short separation distance (10–20 mm) after an 8–14 min run on an agarose gel.
Figure 1. High-throughput agarose gel electrophoresis (FastRuler High Range DNA Ladder).
Tip 2: Choosing the optimal agarose gel concentration
Agarose concentration has a big impact on the quality of separation of your sample or ladder on a gel. The longer the DNA fragment being analyzed, the lower the agarose gel concentration needed (see Table 1 below).
Agarose concentration affects the range of efficient seperation
|Agarose concentration (%)||Range of efficient separation|
Figure 2. (A) Agarose concentration can affect resolution of DNA ladders. (B) Band resolution is improved with an agarose concentration properly selected for the range of fragments to be analyzed.
Tip 3: Choosing the optimal running buffer for electrophoresis
Should you use TAE or TBE running buffer?
- Longer fragments are better resolved when TAE buffer is used (usually for fragments >1 kb)
- Compatible with enzymatic reactions
- Recommended for preparative gel electrophoresis
- Commonly used for better separation of small DNA fragments
Figure 3. Selection of the optimal running buffer. Linear double-stranded nucleic acid fragments migrate approximately 10% slower in TBE buffer.
Tip 4: Choosing a proper sample loading dye/buffer
The sample loading buffer serves two purposes:
- It provides a visible dye that enables you to see the sample during gel loading; it also forms the dye front that allows you to see how far the gel run has progressed.
- It contains a high percentage of glycerol, which makes the sample heavier than the running buffer so that it sinks to the bottom of the well, preventing diffusion into the running buffer.
Once you’ve chosen a loading buffer, be sure to use the same one for both the DNA samples and the ladders. Avoid masking your bands of interest with the tracking dyes that are present in loading buffers. For example, 6X Orange DNA loading buffer contains Orange G (migrates similar to 50 bp DNA) and xylene cyanol (migrates similar to 4,000 bp DNA) tracking dyes. So while this loading buffer is generally very suitable for electrophoresis of small fragments, bands of ~50 bp in size may not be visible due to masking by the Orange G.
Tip 5: Choosing the optimal amount of sample for electrophoresis
- 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.
- Too little DNA can be hard to detect on a gel, particularly the smaller bands that may appear faint.
Ensure the amount of DNA loaded into each well is at least 20 ng per band if the gel is stained using ethidium bromide (EtBr) or SYBR Safe DNA Gel Stain. SYBR Gold Nucleic Acid Gel Stain is more sensitive than EtBr or SYBR Safe DNA Gel Stain. If using SYBR Gold Nucleic Acid Gel Stain, the DNA loaded into each well should be at least 1 ng per band.
Figure 5. The effect of sample concentrations on migration patterns.
Tip 6: Choosing the optimal gel size
- For small gels: 8 x 10 cm gels (mini gels) are commonly used, and documentation of gels of this size is very convenient. The volume of agarose solution for mini gels is typically 30–50 mL.
- For larger gels: Larger gels are used for applications such as Southern and northern blots. The volume of agarose solution for these gels should be about 250 mL.
Tip 7: Avoiding the “smiling” effect
The main causes of bands “smiling” on a gel are:
- Uneven heating of the gel across different lanes. This is usually caused by high voltage. To avoid this, the user can run the gel slowly (reduce the voltage) so that the 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 with your electrophoresis tank.
Figure 6. The “smiling” effect on a gel.
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 running buffer can cause poor resolution, band distortion, or even melting of the gel. However, excess running buffer will also decrease DNA mobility and cause band distortion.
Figure 7. The volume of running buffer can affect DNA mobility and band resolution.
For Research Use Only. Not for use in diagnostic procedures.