Choosing the right electrophoresis products for your nucleic acid analysis workflow is critical to the success of your experiment. This article is a quick guide on how to improve gel electrophoresis results.
Tip 1: Choosing the right ladder
Ladder selection for sizing PCR products or high-throughput gels is an important step in molecular biology experiments. The Thermo Fisher Scientific FastRuler DNA ladders are designed for fast separation and short migration distances and can be a great option for these applications. Here are some guidelines for choosing the right ladder:
- 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).
Click image to enlarge
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.
Tip 2: Choosing the optimal agarose gel concentration
Agarose concentration can have a big impact on the quality of separation of sample or ladder on a gel. Lower agarose gel concentration is suitable for analyzing longer DNA fragments, and vice versa (Figure 2).
Click image to enlarge
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.
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.
Click image to enlarge
Figure 3. TAE versus TBE running buffers in DNA electrophoresis. Linear double-stranded nucleic acid fragments migrate approximately 10% slower in TBE buffer, so TAE is best used to differentiate smaller DNA fragments while TBS is best used to differentiate heavier or longer DNA fragments.
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
The sample loading buffer serves two purposes in DNA gel electrophoresis:
- It contains a dye that renders DNA the sample visible; the migration of the dye front indicates how far the gel run has progressed
- It contains a high percentage of glycerol, which makes the sample heavier than the running buffer; this allows the sample to sink to the bottom of the well and helps prevent the sample from diffusing into the running buffer
Click image to enlarge
Figure 4. Choosing the best sample loading buffer. Sample loading buffers contain dyes that migrate at different DNA lengths. Xylene cyanol migrates like a shorter DNA fragment while Orange G migrates similar to a heavier DNA fragment. Bromophenol blue migrates between xylene cyanol and Orange G.
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
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.
Click image to enlarge
- 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.
Tip 6: Choosing the optimal gel size
- For small gels: 8 x 10 cm gels—also called mini gels—are commonly used. Documentation for these gels is conveniently provided. The volume of agarose solution for mini gels is typically 30–50 mL.
- For larger gels: Larger gels are used in applications such as southern and northern blotting. The volume of agarose solution for these gels should be about 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:
Click image to enlarge
- 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.
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.
Click image to enlarge
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.