Gel Electrophoresis Calculators

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Gel electrophoresis separates molecules (DNA, RNA, proteins) by size as they migrate through a gel matrix under an electric field. Smaller molecules move faster through the gel's porous network; larger molecules move slower. DNA and RNA are separated on agarose gels (0.5–3% for different size ranges); proteins are typically separated on polyacrylamide gels (SDS-PAGE). After electrophoresis, nucleic acids are visualized with ethidium bromide, SYBR Green, or GelRed fluorescent dyes; proteins with Coomassie or silver stain. Molecular weight markers (ladders) run alongside samples to estimate fragment or protein size.

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Agarose Gel Electrophoresis (DNA/RNA)

Agarose concentration determines separation range: 0.5–0.8%: large DNA (5–50 kb); 1.0%: standard (0.3–10 kb); 1.5–2.0%: small fragments (0.1–2 kb); 3%: very small (<200 bp). Buffer: TAE (Tris-acetate-EDTA) or TBE (Tris-borate-EDTA). Loading dye contains: glycerol (densifies sample for loading), tracking dye (bromophenol blue ≈ 300 bp; xylene cyanol ≈ 4 kb in TBE). Voltage: 5–10 V/cm (higher voltage = faster but lower resolution and smearing).

Staining DNA Gels

  • Ethidium bromide (EtBr): intercalates into DNA; UV fluorescence (302 nm excitation, orange emission); toxic/mutagenic → replaced in many labs
  • SYBR Safe / GelRed: safer alternatives; similar sensitivity to EtBr
  • Post-stain: gel placed in staining solution after running; pre-stain: dye added to gel before pouring

Reading Agarose Gel Results

Bands closer to the wells (negative electrode) = larger fragments. Bands further = smaller. Compare to DNA ladder: 1 kb ladder has bands at 10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250 bp. Smearing: DNA degradation or too much DNA loaded. Multiple bands: multiple products or incomplete digestion. No band in expected position: PCR failed, insufficient DNA, or loading error.

2D Gel Electrophoresis

First dimension: isoelectric focusing (IEF) separates proteins by charge (pI). Second dimension: SDS-PAGE separates by size. Resolves thousands of proteins simultaneously; used in proteomics.

Glossary

Gel Electrophoresis
Separation of nucleic acids or proteins by size through a porous gel matrix under electric field; smaller molecules migrate faster; agarose for DNA/RNA, polyacrylamide for proteins.
DNA Ladder
A mixture of DNA fragments of known sizes run alongside samples to calibrate fragment size estimation; common types: 100 bp ladder, 1 kb ladder; essential reference for gel interpretation.
Ethidium Bromide (EtBr)
A fluorescent DNA intercalating dye used to visualize nucleic acids in agarose gels under UV light; mutagen/carcinogen — replaced in many labs by safer alternatives (SYBR Safe, GelRed).

Frequently Asked Questions

DNA is negatively charged (phosphate backbone) and migrates toward the positive electrode in an electric field. The agarose gel acts as a molecular sieve — a porous network with interconnected pores. Smaller DNA fragments pass through the pores more easily and migrate faster; larger fragments are impeded and migrate slower. After electrophoresis (typically 30–90 min at 80–120 V), the gel is stained with a fluorescent dye (EtBr, SYBR Green) and visualized under UV light. Fragment sizes are estimated by comparing migration distances to a DNA ladder (mixture of fragments of known sizes).

Agarose concentration determines pore size and the optimal size range for separation: 0.5–0.8%: large DNA fragments (5–50 kb); 1.0%: standard use (0.3–10 kb, covers most PCR products and restriction digests); 1.5–2.0%: small PCR products (100–2000 bp); 2.5–3.0%: very small fragments (<200 bp) or close-in-size bands. Higher % = smaller pores = better resolution of small fragments but poor resolution of large DNA. Choose concentration based on expected fragment sizes: if running a restriction digest producing bands from 200 bp to 8 kb, use 1.0–1.5% for reasonable resolution across the range.

Choose a DNA ladder whose size range covers your expected fragments. Common ladders: 100 bp ladder (100–1000 bp in 100 bp increments) for PCR products; 1 kb ladder (250 bp–10 kb) for restriction digests and larger PCR products; Quick-Load Purple 1 kb Plus Ladder (NEB) has a bright 1.5 kb reference band for quick orientation. Reading: load 0.5–1 μg of ladder per lane. After staining, identify size standards by their migration position. The 1 kb ladder band appears at the mid-gel position for a 1% gel run at 90 V for 45 min — use this as a reference point, then estimate other bands by interpolation.

Multiple bands when expecting one: (1) Non-specific amplification — PCR primers annealing to off-target sequences; optimize annealing temperature or redesign primers. (2) Incomplete restriction digestion — partial digest due to insufficient enzyme, time, or buffer problems; rerun with fresh enzyme. (3) Concatemers or primer dimers — short (~100 bp) bright bands at the bottom of the lane; improve PCR conditions. (4) Genomic DNA contamination — in RT-PCR, a second band at a different size (usually larger due to intron) indicates genomic DNA; use no-RT control to confirm and treat RNA with DNase. (5) Multiple alleles or splice variants — expected in heterozygous organisms or when studying alternatively spliced mRNA.