Agarose gel electrophoresis is the workhorse technique for separating, identifying, and purifying DNA and RNA fragments by size. It is simple in principle — charged nucleic acids migrate through a porous agarose matrix under an electric field — but the details matter. Gel percentage, buffer choice, voltage, loading dye, and staining method all affect resolution, band sharpness, and whether you can trust your results. This protocol covers everything from pouring to imaging.

How Agarose Gel Electrophoresis Works

Agarose, a polysaccharide derived from seaweed, forms a porous gel when dissolved in buffer and cooled. When an electric field is applied, negatively charged DNA migrates toward the positive electrode (anode). Smaller fragments migrate faster through the gel pores, producing size-based separation. The relationship between migration distance and log(fragment size) is approximately linear within the effective resolution range of a given gel percentage.

Choosing the Right Agarose Percentage

The agarose concentration determines the pore size of the gel matrix, which dictates the range of fragment sizes you can resolve:

| Agarose % | Effective Resolution Range | Common Use | |-----------|---------------------------|------------| | 0.5% | 1–30 kb | Large genomic fragments, BAC digests | | 0.8% | 0.5–12 kb | General cloning, restriction digests | | 1.0% | 0.25–10 kb | Standard PCR products, plasmid digests | | 1.5% | 0.1–3 kb | Small PCR products, multiplex PCR | | 2.0% | 0.05–2 kb | Very small fragments, primer dimers | | 3.0% | 0.01–1 kb | Microsatellites, SNP genotyping (consider MetaPhor agarose) |

For most routine work — checking PCR products, verifying restriction digests, screening clones — a 1% gel is the default.

Materials and Reagents

Agarose: Molecular biology grade (e.g., Sigma Cat# A9539 or Bio-Rad Cat# 1613100). Use low-melting-point agarose (e.g., NuSieve GTG, Lonza) if you plan to excise and purify bands.
Running buffer: 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH ~8.3) or 1× TBE (89 mM Tris-borate, 2 mM EDTA, pH ~8.3).
DNA stain: Ethidium bromide (0.5 µg/mL), SYBR Safe (Thermo Fisher, 1×), or GelRed (Biotium, 1×).
Loading dye: 6× concentrate containing bromophenol blue and/or xylene cyanol with glycerol or Ficoll.
DNA ladder: Select based on your expected fragment sizes. Common choices: 1 kb Plus DNA Ladder (Thermo Fisher, Cat# 10787018), 100 bp DNA Ladder (NEB, Cat# N3231).
Gel casting tray, comb, electrophoresis tank, power supply.

TAE vs. TBE: Which Buffer?

| Factor | TAE | TBE | |--------|-----|-----| | Resolution of large DNA (> 4 kb) | Better | Adequate | | Resolution of small DNA (< 1 kb) | Adequate | Better | | Gel extraction compatibility | Yes (low ionic strength) | No (borate inhibits ligase) | | Buffer exhaustion | Faster (recirculate or replace) | Slower | | Cost | Lower | Slightly higher |

Rule of thumb: Use TAE if you might gel-extract bands. Use TBE for analytical gels where you need sharp small fragments.

Step-by-Step Agarose Gel Protocol

Step 1: Prepare the Gel

Weigh agarose: for a 1% gel with 100 mL volume, weigh 1.0 g agarose.
Add agarose to 100 mL of 1× TAE or TBE buffer in an Erlenmeyer flask.
Microwave in 30-second intervals, swirling between pulses, until the agarose is completely dissolved and the solution is clear. Watch for boil-overs. Total time: 1–3 minutes depending on volume and microwave power.
Cool to approximately 55–60°C (you should be able to hold the flask comfortably). This takes 5–10 minutes on the bench.
Add DNA stain (if using pre-cast staining):
- Ethidium bromide: 5 µL of 10 mg/mL stock per 100 mL gel (final: 0.5 µg/mL)
- SYBR Safe: 10 µL of 10,000× stock per 100 mL gel
- GelRed: 5 µL of 10,000× stock per 100 mL gel
Swirl gently to mix (avoid creating bubbles).
Pour into the casting tray with the comb(s) in place. Pop any bubbles with a pipette tip.
Allow to solidify at room temperature for 20–30 minutes (the gel turns opaque when set).

Step 2: Load Samples

Remove the comb carefully by pulling straight up with a gentle rocking motion.
Place the gel in the electrophoresis tank and fill with 1× running buffer until the gel is submerged by 2–3 mm.
Mix your DNA samples with 6× loading dye (1 µL dye per 5 µL sample). Loading dye serves two purposes: it makes the sample sink into the well, and the tracking dyes indicate migration progress.
Load samples into wells using a micropipette. Typical volumes: 5–20 µL for mini gels, 20–40 µL for midi gels.
Always load a DNA ladder in at least one lane. Load the amount recommended by the manufacturer (usually 0.5–1 µg for sharp, quantifiable bands).

Step 3: Run the Gel

Connect electrodes: DNA runs toward the red (positive) electrode. Place the wells at the black (negative) end.

Voltage guidelines:

Standard analytical gel: 5–8 V/cm (measure electrode-to-electrode distance, not gel length). For a 10 cm electrode gap, use 50–80 V.
Quick check (sacrifice resolution): up to 10 V/cm
Large fragments or pulsed-field gels: 1–3 V/cm

Running time depends on voltage and the distance you need the fragments to migrate. Typical: 30–60 minutes at 100 V for a mini gel.

Monitor the tracking dyes:

Bromophenol blue migrates with ~300 bp fragments in a 1% gel
Xylene cyanol migrates with ~4 kb fragments in a 1% gel

Stop the run when the leading dye is 1–2 cm from the bottom of the gel.

Step 4: Stain (If Not Pre-Cast)

If you did not add stain to the gel before casting, use post-staining:

Ethidium bromide: Soak gel in 0.5 µg/mL EtBr in water or running buffer for 15–30 minutes. Destain in water for 10 minutes to reduce background.
SYBR Safe or GelRed: Soak in 1× solution for 30 minutes. No destaining needed.

Post-staining can give more uniform staining and lower background than pre-casting, especially for thick gels.

Step 5: Image the Gel

EtBr-stained gels: Visualize on a UV transilluminator (302 nm) or gel documentation system. Use a UV filter or amber filter for photography.
SYBR Safe/GelRed gels: Use blue-light transilluminator (recommended — less DNA damage for gel extraction) or UV.
For gel extraction: Minimize UV exposure time to < 30 seconds. Extended UV causes pyrimidine dimers that reduce cloning and transformation efficiency.

Optimizing Band Resolution and Sharpness

Fuzzy or Smeared Bands

Overloaded gel: Reduce DNA amount. For analytical purposes, 50–200 ng total DNA per lane is sufficient.
Degraded DNA: Run a fresh extraction. DNA with nicks runs as a smear rather than discrete bands.
Too high voltage: Excessive heat causes band diffusion. Reduce voltage to 5 V/cm.
Old buffer: Replace TAE buffer — it exhausts faster than TBE, especially at high voltage. Recirculate or refresh every 2–3 runs.

Curved or Smiling Bands

Uneven gel thickness: Ensure the casting tray is level.
Excessive heat in center lanes: Run at lower voltage or use a tank with buffer recirculation.
Overloaded outer lanes: Edge effects are worsened by high sample volumes.

Bands Running at Wrong Size

Supercoiled plasmid DNA: Undigested plasmid migrates differently than its linear size suggests. Supercoiled DNA runs faster than linear, which runs faster than relaxed circular (nicked).
Secondary structure in single-stranded DNA/RNA: Denature with formamide loading buffer or run under denaturing conditions (formaldehyde gel for RNA).
Wrong ladder used: Confirm the ladder matches your expected size range.

No Bands Visible

DNA concentration too low: Load more DNA or use a more sensitive stain. EtBr detection limit is ~10 ng per band; SYBR Safe can detect ~1 ng.
DNA ran off the gel: Check tracking dyes. If they are gone, you ran too long.
Wrong staining protocol: Ensure correct excitation wavelength for your stain.
Reversed electrodes: DNA migrates toward positive (red). Check your connections.

Gel Extraction Tips

If you need to purify a band for downstream cloning or sequencing:

Use a low-melting-point agarose gel or standard agarose with a commercial gel extraction kit (QIAquick Gel Extraction Kit, Qiagen Cat# 28704; or Monarch DNA Gel Extraction Kit, NEB Cat# T1020).
Excise the band with a clean scalpel under blue light (not UV if possible).
Minimize the gel slice volume — excess agarose reduces recovery.
Melt the gel slice at 50°C in the provided solubilization buffer. Follow kit instructions for column binding, washing, and elution.
Elute in warm (55°C) nuclease-free water or EB buffer for maximum recovery. Typical yield: 50–80% of input DNA.

Running RNA on Agarose Gels

RNA analysis requires denaturing conditions to eliminate secondary structure. Use a 1–1.2% agarose gel prepared with 1× MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) and 2.2 M formaldehyde. Denature RNA samples at 65°C for 5 minutes in formaldehyde loading buffer before loading.

Intact total RNA shows two sharp ribosomal bands (28S and 18S rRNA). The 28S band should be approximately twice the intensity of the 18S band. If both bands are faint or absent, the RNA is degraded.

How LabProtocol.co Can Help

Getting gel electrophoresis conditions right — agarose percentage, buffer choice, voltage, staining method — depends on your specific fragment sizes and downstream goals. LabProtocol.co can generate an optimized gel electrophoresis protocol tailored to your experiment in seconds. Try it free and skip the trial-and-error.

Quick Reference

Default gel: 1% agarose in 1× TAE at 5–8 V/cm for 30–60 minutes.
Use TAE when you might gel-extract bands. Use TBE for sharper analytical resolution.
Pre-cast staining with SYBR Safe or GelRed is safer and more convenient than EtBr.
Load 50–200 ng DNA per lane for clean, quantifiable bands.
Always run a ladder. Always check your electrode orientation. Always photograph your gel before it dries out.