Virtual Agarose Gel Electrophoresis

🧪 1. What is agarose gel electrophoresis?

Agarose gel electrophoresis separates DNA fragments by size. DNA is negatively charged (due to its phosphate backbone), so when an electric field is applied, fragments migrate from the negative electrode (cathode, black) toward the positive electrode (anode, red).

Smaller fragments move through the agarose mesh faster than larger ones, so after the run, fragments are sorted by size — large near the wells, small near the bottom.

Key rule: Migration distance is inversely proportional to log₁₀(bp). Plotting log(bp) vs distance gives a near-straight line — that's how we estimate unknown fragment sizes.

📊 2. Choosing Agarose Concentration

The agarose percentage determines the pore size of the gel — and therefore which fragment sizes it can resolve.

Agarose %	Resolvable size range	Best for
0.5%	1,000 – 30,000 bp	Very large fragments, genomic DNA
0.8%	500 – 10,000 bp	Plasmid digests, PCR products >1 kb
1.0%	250 – 10,000 bp	General purpose — most common
1.2%	150 – 6,000 bp	Mid-range PCR products
1.5%	100 – 3,000 bp	Small PCR products, genotyping
2.0%	50 – 1,000 bp	Small fragments, close-size resolution
3.0%	10 – 500 bp	Very small fragments (e.g. miRNA)

Tip: If two bands are close in size (ratio < 1.2×), increase the agarose percentage and/or run longer to resolve them.

⚡ 3. Voltage, Current & Run Time

The electric field strength determines how fast DNA migrates:

Voltage (V): Usually 1–10 V/cm of gel length.
• 5 V/cm is standard for most applications.
• > 10 V/cm → gel heating, band smearing, distortion.
Current (mA): Proportional to voltage and buffer ionic strength.
Run time: Longer = better resolution (up to a point).

Voltage	Speed	Resolution	Comment
Low (≤ 50 V)	Slow	Excellent	Best for close fragments
Medium (50–120 V)	Standard	Good	Recommended range
High (120–200 V)	Fast	Fair	Acceptable for quick checks
Very high (> 200 V)	Very fast	Poor	Overheating, smearing, melting

Warning: Never run a gel > 5 V/cm for extended periods without cooling — it can melt or distort the agarose.

🧴 4. Buffer Systems

Buffer	Composition	Strengths	Weaknesses
TAE	40 mM Tris-acetate, 1 mM EDTA, pH 8.0	Best for large DNA, long runs	Lower buffering capacity; must recirculate for long runs
TBE	89 mM Tris-borate, 2 mM EDTA	Higher buffering capacity; best for small fragments	Borate can inhibit downstream enzymatic reactions
0.5× TBE	Diluted TBE	Routine use; cheaper	Slightly lower resolution
SB	Sodium borate	Cheap, allows high voltage without overheating	Less common; not for all applications
LB	Lithium borate	High-resolution at high voltage	Specialised use

💡 5. DNA Stains

Stain	Colour under UV	Sensitivity	Safety
Ethidium bromide (EtBr)	Orange-red	~0.5 ng / band	Mutagenic — handle carefully
SYBR Green	Bright green	~0.1 ng / band (most sensitive)	Irritant; safer than EtBr
GelRed	Green (under UV) / red (under gel-doc)	~0.1 ng / band	Safer alternative to EtBr
SYBR Safe	Blue-green	~0.5 ng / band	Very safe — blue-light excitable

Band intensity is roughly proportional to the mass of DNA in the band (not copy number). A 3 kb band has 3× the mass of a 1 kb band at equal molarity, so it will appear brighter.

📏 6. DNA Markers / Ladders

A DNA ladder (marker) is a mixture of fragments of known sizes, used as a reference to estimate the size of unknown bands.

Ladder	Range	Best for
100 bp ladder	100 bp – 1.5 kb	Small PCR products
1 kb Plus	100 bp – 10 kb	General purpose (wide range)
λ DNA / HindIII	125 bp – 23 kb	Large fragments, plasmid digests
50 bp ladder	50 bp – 1 kb	Very small fragments

How to use a ladder: Draw a line from each ladder band across the gel; the unknown bands between two ladder bands have sizes between those two values. For precise size estimation, plot log₁₀(bp) vs migration distance on the ladder and interpolate.

🔍 7. How to Read Your Gel Image

The virtual gel generated by this tool contains:

Lane 1 — the DNA ladder (with size labels on the right).
Lanes 2+ — your samples, one lane per line you entered.
Bright bands — larger fragments (they bind more stain per band).
Dim bands — smaller fragments or low-mass bands.
Wells at the top — where samples were loaded.
Migration arrow — direction of DNA movement (top → bottom).

Interpreting your results

Observation	Interpretation
Single sharp band per sample	Pure, single-size DNA — good prep.
Multiple bands in a lane	Mixed sample, digest, or PCR with non-specific products.
Smear (continuous streak)	Degraded DNA, overloaded lane, or too-high voltage.
Band at the well	Very large DNA that hasn't entered the gel (e.g. genomic DNA).
Band runs ahead of the dye front	Small fragments (< 100 bp) — use a higher gel %.
Curved / smeared bands	Overheating, uneven gel, or buffer depletion.

Size estimation: Compare your unknown band's vertical position against the ladder. The band between the 1 kb and 2 kb ladder bands has a size between 1 and 2 kb; closer to 1.5 kb if it's near the middle.

🛠️ 8. Common Problems & Troubleshooting

Problem	Likely cause	Fix
No bands visible	Forgot stain / no DNA loaded / UV off	Add stain, reload sample, check UV
Bands very faint	Low DNA amount / poor stain sensitivity	Load more DNA, use SYBR Green
Bands smeared	Too much DNA, high voltage, degraded sample	Dilute sample, reduce V, re-prep DNA
Bands distorted / smiling	Overheating or uneven electric field	Lower voltage, use thinner gel, cool buffer
Poor resolution of close bands	Wrong gel % / short run	Increase gel %, run longer at lower V
Ladder not visible	Ladder too dilute / stain issue	Load fresh ladder, ensure stain
Bands in all lanes at same position	Contamination (common in PCR)	Use fresh reagents, negative control
Bands run in wrong direction	Electrodes reversed	DNA migrates toward + (red); check leads

Critical safety notes: EtBr is a mutagen — always wear gloves. UV light damages eyes and skin — use a face shield. Electricity + liquid buffer = risk of electric shock — turn off the power supply before opening the lid.

Pre-run checklist

✔ Gel % matches fragment size range
✔ Buffer freshly prepared and at correct dilution
✔ Gel fully submerged (2–3 mm above surface)
✔ Samples mixed with loading dye (adds density + tracking dye)
✔ Ladder loaded in lane 1
✔ Electrodes connected correctly (DNA runs toward +)
✔ Voltage ≤ 5 V/cm for best resolution
✔ Stain added (in-gel or post-stain)

🧪 Virtual Agarose Gel Electrophoresis Simulator

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