Introduction
Agarose gel electrophoresis is likely one of the first experiments you performed in the lab. Today, let's take a deep dive into this essential technique—from the underlying principles to practical tips and troubleshooting strategies.
I. Principle Section
1) What is the principle of agarose gel electrophoresis?
Agarose gel electrophoresis is a method that uses agarose as a supporting matrix and is widely used for DNA fragment recovery, size separation, and validation of recombinant DNA or plasmid digestion.
How it works: Agarose forms a porous network structure that exerts resistance as DNA migrates through it. Because DNA fragments differ in size and charge, they move at different speeds under an electric field—migrating from the negative to the positive electrode—allowing for size-based separation.
II. Practical Section
1) How to choose the right agarose concentration?
Answer: Choose based on the size of the nucleic acid fragments you're working with. Refer to the standard concentration chart for preparation guidance.
2) How to select a nucleic acid stain?
Answer: Common nucleic acid stains on the market include EB, GoldView, and GelRed/GelGreen.
- EB (Ethidium Bromide): Intercalates between DNA base pairs, with peak UV absorption at 300 and 360 nm and orange-red fluorescence emission at 590 nm. Advantages include high sensitivity and low cost—but it's a mutagen and toxic to humans.
- GoldView: Primarily composed of acridine orange, stains both DNA and RNA and displays different fluorescence colors. Its excitation peak is 492 nm, and emission peaks are 530 nm (DNA) and 640 nm (RNA). Works well for large fragments but not ideal for small fragments (<500 bp). Fluorescence fades quickly under UV light and is not suitable for gel recovery.
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GelRed/GelGreen: Safer alternatives as they do not penetrate cell membranes.
- GelRed emits red fluorescence (similar to EB).
- GelGreen emits green fluorescence (similar to SYBR® Green or SYBR® Safe).
- Both are UV-compatible, with GelGreen also suitable for blue-light transilluminators.
3) Key tips for running a successful gel electrophoresis:
- Do not overfill the gel flask—keep volume under 50% to avoid overflow during heating.
- Ensure complete melting of agarose during heating. Cover with sealing film (not airtight) to reduce evaporation.
- Pour the gel at the right temperature—not too hot to avoid bubbles.
- Stain addition: You may pre-mix stain into the gel (convenient) or post-stain using a soaking method.
III. Troubleshooting Section
1) Bands are not well separated?
Possible causes:
- Electrophoresis time too short—try running longer.
- Gel concentration incorrect—check for weighing or evaporation errors.
- High salt concentration in the sample—can affect mobility.
2) No bands visible after electrophoresis?
Two scenarios:
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Neither marker nor sample shows bands:
- Dye not added or degraded
- Incorrect electrophoresis parameters
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Marker shows bands, sample doesn’t:
- PCR failed to amplify target—optimize PCR conditions
- Loading buffer not added properly
- Nucleic acid extraction failed or concentration too low
3) Smiling or smearing bands?
Likely due to issues with the gel, stain, sample, or electrophoresis conditions:
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Gel-related:
- Incomplete agarose melting or insufficient gel setting time leads to uneven pore sizes and uneven migration.
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Stain-related:
- High temperature during staining can damage dye structure. Add stain when gel cools to 40–50°C.
- Uneven stain distribution causes migration differences.
- Solution: Mix thoroughly after adding stain or use post-staining method.
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Sample-related:
- Smiling bands: Caused by overloading (usually >500 ng). For PCR, enzyme digestion, or markers, 3–5 µL is enough.
- Smearing (tailing): May be due to degradation. Handle samples with gloves and RNase/DNase-free tools—especially critical for RNA.
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Electrophoresis-related:
- High voltage (>150V) can cause smearing. Recommended: 110–130V.
- Also check the pH and ionic strength of the running buffer—always use freshly prepared buffer.
4) Sample stuck in the wells?
Potential causes:
- Protein or cell debris in the sample crosslinked with DNA/RNA
- Overloading
- Incorrect voltage or buffer composition
5) Faint bands?
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Large fragment bands faint: Large DNA fragments bind stain less efficiently.
Solution: Add more stain, reduce loading volume. -
Small fragment bands faint: May be affected by stain migration.
Solution: Consider using post-staining method.
Guidelines for the selection of related products
Conventional PCR |
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Specification |
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Amplification Length |
≤10-15 kb |
≤5 kb |
≤5 kb |
≤4 kb |
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Extension Time |
1-10 sec/kb |
30 sec/kb |
30 sec/kb |
30 sec/kb |
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Product End Structure |
3’-dA |
3’-dA |
3’-dA |
3’-dA |
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Annealing Temperature |
60℃ |
Tm-(2~5)℃ |
Tm-(2~5)℃ |
Tm-(2~5)℃ |
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GC Compatibility Range |
30-70% |
40-70% |
40-70% |
30-70% |
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5'-3' Exonuclease Activity |
Present |
Present |
Present |
Present |
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Colony PCR |
Suitable |
Suitable |
Suitable |
Suitable |
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Gene Identification |
Suitable |
Suitable |
Suitable |
Suitable |
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Multiplex PCR |
Not Suitable |
Not Suitable |
Not Suitable |
3-4 plex PCR |
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Electrophoresis Indicator |
Purple-red |
Blue |
Colorless |
Blue |
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Hot Start |
Hot Start |
Not Hot Start |
Not Hot Start |
Hot Start |
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Pre-mix/Kit |
Pre-mix |
Pre-mix |
Pre-mix |
Pre-mix |
Direct PCR |
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Specification |
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Product Type |
Mouse Direct Amplification |
Blood Direct Amplification |
Plant Direct Amplification |
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Amplification Length |
≤1 kb |
≤8 kb |
≤1 kb |
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Extension Time |
30 s/kb |
3-5 s/kb for ≤2 kb, |
60 s/kb |
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10 s/kb for ≤8 kb |
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Sample lysis time |
15 min |
0-3 min |
0-10 min |
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Product End Structure |
Blunt End |
Blunt End |
Blunt End |
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Annealing Temperature |
Tm-(2~5)℃ |
Tm-(1~2)℃ |
Tm-(2~5)℃ |
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GC Compatibility Range |
30-70% |
30-75% |
40-65% |
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5'-3' Exonuclease Activity |
√ |
√ |
√ |
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Gene Identification |
√ |
√ |
√ |
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Multiplex PCR |
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3-4 plex |
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Direct Sample Amplification |
√ |
√ |
√ |
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Electrophoresis Indicator |
Blue |
Colorless |
Blue |
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Hot Start |
√ |
√ |
√ |
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Pre-mix/Kit |
Kit (with Mix) |
Kit (with Mix + Buffer) |
Kit (with Mix) |
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Suitable Organisms |
Mouse, Rat |
Human, Mouse, Goat, Chicken, Pig, etc. |
Rice, Corn, Tobacco, Rapeseed, Wheat, Soybean, etc. |
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Suitable Tissue/Material |
Tail, Ear, Toe (with muscle), and other organs |
Fresh blood with EDTA, Heparin, Citrate, etc., refrigerated (frozen) blood, commercial dry blood spots |
Young leaves, old leaves, seedlings, young stems |
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Sample input |
Tissue: 5-10 mg; Tail: 1-5 mm |
Whole blood: 0.5%-20%, dry blood spot: 1 mm² |
Leaf: 1-10 mm, Seed: 1-3 mm |
High-Fidelity PCR |
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Specification |
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Amplification Length |
≤10 kb gDNA, ≤10 kb cDNA, ≤16 kb λDNA |
≤10 kb gDNA, ≤10 kb cDNA, ≤13 kb λDNA |
≤10 kb gDNA, ≤10 kb cDNA, ≤13 kb λDNA |
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Extension Time |
5 sec/kb |
30 sec/kb |
30 sec/kb |
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Fidelity (Taq) |
83× |
83× |
83× |
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Product End Structure |
Blunt End |
Blunt End |
Blunt End |
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Annealing Temperature |
60℃ |
68℃ |
68℃ |
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GC Compatibility Range |
20-80% |
30-60% |
30-60% |
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5’-3’ Exonuclease Activity |
Absent |
Absent |
Absent |
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Electrophoresis Indicator |
Blue |
Colorless |
Blue |
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Single Enzyme/Pre-mix |
Pre-mix |
Single Enzyme |
Pre-mix |
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Tolerance |
/ |
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Blood, Mouse Tissue Lysate |
Nucleic Acid Electrophoresis |
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Product Category |
Cat NO. |
Product Name |
Application |
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Agarose |
Agarose |
Routine nucleic acid electrophoresis |
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High Sieving Agarose (PCR Grade) |
Suitable for separating DNA fragments of 20 bp-800 bp, comparable to polyacrylamide gel |
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Agarose Tablets (0.5 g/tablet) |
Convenient for routine agarose applications |
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Nucleic Acid Dye |
YeaRed Nucleic Acid Gel Stain (10,000× in Water) |
Water-soluble, with spectral properties similar to EB, excitable at 300 nm UV light |
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DNA Marker |
GoldBand 1 kb DNA Ladder |
250-12000 bp (13 bands) |
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GoldBand 50 bp DNA Ladder |
50-1000 bp (14 bands) |
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GoldBand 100bp DNA Ladder |
100-1500 bp (12 bands) |
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GoldBand 100 bp plus DNA Ladder |
100-3000 bp (14 bands) |
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GoldBand 200 bp DNA Ladder |
200-5000 bp (12 bands) |
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GoldBand 500 bp DNA Ladder |
500-5000 bp (8 bands) |
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GoldBand DL2000 DNA Marker |
100-2000 bp (6 bands) |
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GoldBand DL5000 DNA Marker |
100-5000 bp (9 bands) |
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GoldBand DL10,000 DNA Marker |
100-10000 bp (10 bands) |
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GoldBand Full-Scale DNA Ladder |
100-12000 bp (20 bands) |
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GoldBand DL15000 DNA Marker |
250-15000 bp (7 bands) |
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