Understanding Gel Electrophoresis

ENTM201L - General Entomology Laboratory | UC Riverside

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Understanding Gel Electrophoresis

Principles of DNA Separation and Visualization

ENTM201L - Lab Theory

Why Gel Electrophoresis?

After PCR amplification of the mosquito COI gene in Lab, you face several critical questions:

Gel electrophoresis answers all these questions simultaneously by physically separating DNA fragments by size and visualizing them with fluorescent dyes. This 50-year-old technique remains the gold standard for PCR quality control because it is simple, reliable, inexpensive, and provides visual confirmation that other methods cannot match.

The Fundamental Principle: DNA Migration in Electric Fields

DNA is a Charged Molecule

DNA carries a negative charge due to phosphate groups in its sugar-phosphate backbone. Each nucleotide contributes one phosphate (PO₄⁻), meaning:

Key Insight: All DNA molecules have the same charge-to-mass ratio. A 100 bp fragment has 200 negative charges and weighs 65 kDa. A 1000 bp fragment has 2,000 negative charges and weighs 650 kDa. The ratio is constant: 2 charges per 650 Da.

Electrophoresis: Movement Through an Electric Field

When DNA is placed in an electric field:

1. Negative DNA is attracted to the positive electrode (anode)

2. DNA migrates from negative (cathode) toward positive (anode)

3. Electric field strength determines migration speed

Without agarose gel, all DNA would migrate at the same speed (same charge-to-mass ratio). The gel provides a molecular sieve that separates by size.

Agarose Gel as a Molecular Sieve

Agarose is a polysaccharide extracted from seaweed that forms a porous matrix when dissolved in buffer and cooled. Structure: How it separates DNA:
DNA Fragment SizeMigration Behavior
Small (100-500 bp)Easily snakes through pores; fast migration
Medium (500-5,000 bp)Moderate difficulty threading through pores
Large (10,000+ bp)Must reptate (snake-like motion); slow migration
Very large (>50 kb)Barely enters gel; gets stuck in pores
The Physics: Small DNA fragments experience less friction as they navigate the gel matrix. Large fragments encounter more pore walls, slowing their progress. The result is size-based separation.

Migration Distance is Proportional to Log(Size)

Mathematical relationship: 
Distance migrated ∝ -log₁₀(fragment size in bp)

This means:

Practical consequence: DNA ladders have evenly spaced bands by molecular weight, but on the gel they appear logarithmically compressed, with large fragments bunched together near the wells.

Agarose Gel Preparation

Selecting Agarose Concentration

The optimal agarose concentration depends on the size range of DNA you expect to see.

Agarose %Effective RangeApplicationPore Size
0.5%1-30 kbGenomic DNA, large plasmidsVery large
0.7%800 bp - 12 kbPlasmids, larger PCR productsLarge
1.0%500 bp - 10 kbStandard PCR productsMedium
1.5%200 bp - 3 kbSmall PCR products, primersSmall
2.0%50-500 bpGenotyping, primer dimersVery small
For ENTM201L COI PCR (712 bp): 1% agarose is optimal. Why?

The Chemistry of Gel Formation

Agarose gelation is temperature-dependent:

1. Heating (90-100°C in microwave):

- Agarose powder dissolves in buffer

- Polysaccharide chains fully dissociated

- Solution is clear and viscous

2. Cooling (60-70°C):

- Still liquid; safe to pour into tray

- Chains begin to associate

- Can add SYBR Safe dye at this temperature

3. Gelation (40-50°C):

- Chains crosslink through hydrogen bonds

- 3D network forms (sol → gel transition)

- Gel becomes opaque and solid

4. Room temperature (20-25°C):

- Fully solidified gel

- Stable for hours in running buffer

- Can be stored at 4°C for weeks

Critical factors:

- TAE: Lower buffering capacity but better for DNA recovery

- TBE: Higher buffering capacity but borate can inhibit downstream enzymes

- ENTM201L uses TAE (standard for preparative gels)

- Unmixed gel has concentration gradients → uneven migration

Preparing a 1% Agarose Gel (Standard Protocol)

Materials: Steps:

1. Weigh 0.5 g agarose into Erlenmeyer flask

2. Add 50 mL 1× TAE buffer

3. Microwave in 30-second bursts, swirling between, until fully dissolved

4. Cool to ~60°C (can touch flask comfortably)

5. Add 5 µL SYBR Safe, mix gently

6. Pour into tray with comb inserted

7. Wait 20-30 minutes for complete solidification

8. Remove comb, creating wells for sample loading

Safety note: Always wear heat-protective gloves when handling hot agarose. Superheated liquid can cause severe burns.

SYBR Safe DNA Staining

Why We Stain DNA

DNA is colorless and invisible under normal light. To visualize bands, we use fluorescent dyes that bind DNA and emit visible light when illuminated with UV or blue light.

SYBR Safe Chemistry

SYBR Safe is a cyanine dye that binds double-stranded DNA through minor groove binding (different from intercalation). Spectral properties: Binding mechanism:

1. SYBR Safe molecule fits into minor groove of DNA helix

2. Binding restricts molecular rotation

3. Fluorescence quantum yield increases ~100-fold

4. Free dye in gel is essentially non-fluorescent

Selectivity:

SYBR Safe vs. Ethidium Bromide

Historically, ethidium bromide (EtBr) was the standard DNA stain. SYBR Safe has replaced it in most labs due to safety advantages.

PropertyEthidium BromideSYBR Safe
MutagenicityStrong mutagen (intercalates DNA)Non-mutagenic in Ames test
CarcinogenicitySuspected carcinogenNot classified as hazardous
Sensitivity1-5 ng DNA per band1-3 ng DNA per band
Excitation302 nm (UV transilluminator)302 nm UV or 470 nm blue light
Emission590 nm (orange)530 nm (green)
DisposalHazardous waste (costly)Regular chemical waste
CostLow (~$50/10 g)Higher (~$200/mL stock)
Why SYBR Safe is safer: ENTM201L uses SYBR Safe to minimize exposure to mutagenic compounds while maintaining excellent sensitivity.

Detection Methods

UV Transilluminator (302 nm): Blue Light Transilluminator (470 nm):

DNA Ladder: The Molecular Ruler

Purpose of DNA Ladders

A DNA ladder (also called marker or standard) is a mixture of DNA fragments of known sizes that serve as a size reference for unknown samples.

Functions:

1. Size estimation: Compare unknown band to ladder bands

2. Quantification: Compare band intensity to ladder (if known ng amounts)

3. Quality control: Verify gel is running properly

4. Orientation: Confirms DNA migrated toward positive electrode

Common Ladders for PCR Analysis

1 kb DNA Ladder (most common for PCR): Example 1 kb ladder pattern: 
10,000 bp (10 kb) ━━━
 8,000 bp ━━━
 6,000 bp ━━━
 5,000 bp ━━━
 4,000 bp ━━━
 3,000 bp (3 kb) ━━━━━ ← Reference band (brightest)
 2,000 bp ━━━
 1,500 bp ━━
 1,000 bp (1 kb) ━━━
 750 bp ━━
 500 bp ━━
 250 bp ━
 100 bp ━

Interpreting Ladder for COI PCR

Expected COI product: 712 bp (using AU-COI primers) Reading the gel:

1. Locate 1 kb (1,000 bp) band on ladder

2. Locate 500 bp band on ladder

3. COI band (712 bp) should fall between these two, closer to 750 bp

4. Distance: ~30% from 1 kb toward 500 bp

Quantification (if ladder has mass data):

- Estimate: Your sample has ~200 ng total DNA in that band

- If you loaded 5 µL from 25 µL PCR: 200 ng ÷ 5 µL × 25 µL = 1,000 ng total

- Concentration: 1,000 ng ÷ 25 µL = 40 ng/µL

This is crude estimation. For accurate quantification, use Qubit after gel extraction.

Loading Dye: Making DNA Visible and Dense

Why Loading Dye is Necessary

Problem 1: DNA solution is less dense than buffer Problem 2: DNA solution is colorless Loading dye solves both problems.

Components of Loading Dye

6× Loading Dye (typical formulation):

1. Glycerol (30-40%) or Ficoll (15%):

- Increases density of sample

- Sample sinks into well and stays there

- Does not migrate during electrophoresis

2. Tracking dyes:

- Bromophenol blue (migrates ~500 bp equivalent)

- Blue color

- Helps you see sample loading

- Tracks migration during run

- Xylene cyanol FF (migrates ~4,000 bp equivalent)

- Light blue/cyan color

- Secondary tracking dye

3. EDTA (optional):

- Chelates Mg²⁺ and Ca²⁺

- Inhibits DNases (protects DNA from degradation)

- Typically 10 mM final concentration

4. Buffer (Tris-HCl):

- Maintains pH

- Prevents DNA degradation

Using Loading Dye

Concentration: Most loading dyes are 6× stock Alternative: Some labs use 1× loading dye pre-diluted Tracking migration:

Running Conditions and Optimization

Voltage, Current, and Time

Electrophoresis parameters:
ParameterTypical ValueEffect
Voltage100-120 VHigher voltage = faster migration
Current50-100 mADepends on buffer and gel size
Power10-15 WDissipated as heat
Time45-60 minUntil adequate separation achieved
Ohm's Law applies: 
V = I × R

Where:

Gel resistance depends on:

Optimizing Run Conditions

Faster runs (higher voltage):

- Denatures DNA (smearing)

- Creates uneven temperature (smile effect)

- Evaporates buffer

- Distorts bands

- Example: 10 cm gel → max 100 V

Slower runs (lower voltage): Standard for COI PCR:

Buffer Circulation and Temperature

Heat dissipation: Solutions:

1. Recirculating buffer: Pump buffer from anode to cathode chamber

2. Ice bath: Place gel box on ice (for long runs)

3. Lower voltage: Reduce heat generation (I²R term decreases)

Buffer depletion:

Interpreting Gel Results

Successful COI PCR (Expected Result)

What you should see: 
Lane: Ladder Sample1 Sample2 Sample3 Blank
 | | | | |
 1kb ━ ━━━ ━━━ ━━━ (nothing)
 750bp ↑ ↑ ↑
 500bp ━ COI at 712 bp
Interpretation: This indicates:

No Visible Band (PCR Failure)

What you see: 
Lane: Ladder Sample Blank
 | | |
 1kb ━ (nothing)(nothing)
 500bp ━
Possible causes:

1. No DNA template:

- Extraction failed

- DNA degraded during storage

- Test: Re-check Qubit concentration

2. PCR inhibitors:

- Proteins, salts, ethanol, melanin

- Test: Dilute DNA 1:5, retry PCR

3. Wrong primers:

- Primers don't match your species

- Primer degraded (freeze-thaw cycles)

- Test: Use positive control DNA

4. Thermocycler error:

- Didn't reach annealing/extension temperature

- Lid heater off (evaporation)

- Test: Run known working sample

5. Reagent failure:

- Polymerase inactive (expired or heat-damaged)

- dNTPs degraded

- Test: Use fresh master mix

Troubleshooting priority:

1. Check template DNA concentration (Qubit)

2. Run positive control PCR

3. Dilute template 1:5 (removes inhibitors)

4. Increase cycles to 40

5. Replace polymerase

Primer Dimers (<100 bp)

What you see: 
Lane: Ladder Sample Blank
 | | |
 1kb ━ ━━━
 500bp ━ COI product
 100bp ━:::(smear/band at ~60 bp)
Cause: Why it happens: 
Forward primer: 5'-GGTCAACAAATCATAAAGATATTGG-3'
 ||||
Reverse primer (3' end): 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

Even 4-5 bp complementarity can nucleate primer dimer formation.

Solutions:

1. Increase annealing temperature (55°C → 58°C)

- Destabilizes weak primer-primer interactions

- Still allows primer-template annealing

2. Decrease primer concentration (0.5 µM → 0.3 µM)

- Less primer-primer collisions

3. Increase template DNA amount

- Favors primer-template over primer-primer

4. Hot-start polymerase

- Q5 is hot-start (inactive until 98°C)

- Should already prevent primer dimers

Impact on sequencing:

Non-Specific Amplification (Multiple Bands)

What you see: 
Lane: Ladder Sample Blank
 | | |
 1kb ━ ━━
 750bp ━━━ ← COI expected
 500bp ━ ━━
 300bp ━
Cause: Why it happens: Solutions:

1. Increase annealing temperature (55°C → 60°C)

- Stringency increases

- Only perfect matches amplify

2. Optimize Mg²⁺ concentration

- Lower Mg²⁺ (1.5 mM → 1.0 mM) increases specificity

3. Redesign primers

- Use more specific primers (longer, or different region)

4. Touchdown PCR

- Start at high annealing temp (65°C), decrease by 1°C each cycle

- Enriches for most specific product

Sequencing strategy:

Smearing (DNA Degradation)

What you see: 
Lane: Ladder Sample Blank
 | | |
 1kb ━::::
 500bp ━:::: ← Continuous smear, no sharp band
 300bp::::
Cause: Sources of DNases:

1. Contaminated water (use molecular biology grade)

2. Dirty pipettes (human skin has DNases)

3. Tube/reagent contamination

4. PCR product degradation (old sample, freeze-thaw)

Solutions:

1. Use filter tips (prevent pipette contamination)

2. Fresh water for PCR master mix

3. Run gel immediately after PCR (don't store product for weeks)

4. Add EDTA to loading dye (inhibits DNases)

Prevention:

Weak Band (Low Yield)

What you see: 
Lane: Ladder Sample Blank
 | | |
 1kb ━━━ ━ (very faint)
 500bp ━
Cause: Solutions:

1. Use more template (5 µL instead of 1 µL)

2. Increase PCR cycles (35-40 instead of 30)

3. Dilute template 1:2 (if inhibitors suspected)

4. Check reagents (fresh polymerase, dNTPs)

Quantification:

Quantifying DNA from Gel Bands

Visual Estimation vs. Fluorometry

Visual comparison to ladder: Example: 
Ladder (100 ng/band) Sample (unknown)
 1 kb ━━━ ━━━━━ ← ~2× brighter
 500 bp ━━━

Estimate: ~200 ng in loaded volume (5 µL from 25 µL)

Total: 200 ng ÷ 5 × 25 = 1,000 ng = 40 ng/µL

Qubit after gel extraction:

Gel Extraction for Sequencing

When to extract:

1. Multiple bands present (isolate correct size)

2. Primer dimers contaminating sample

3. Need precise quantification

4. Non-specific products interfering

Protocol overview:

1. Excise band with clean scalpel (use blue light, not UV)

2. Dissolve agarose in chaotropic buffer (Qiagen QIAquick)

3. Bind DNA to silica column

4. Wash, elute in 30 µL Tris buffer

5. Quantify with Qubit

6. Submit for sequencing

Expected recovery:

Preparing PCR Products for Sanger Sequencing

Quality Requirements

Sequencing facility specifications (e.g., UC Riverside Genomics Core):

1. Concentration: 20-50 ng/µL

2. Purity: A260/A280 >1.8, A260/A230 >2.0

3. Volume: 10 µL PCR product minimum

4. Primer: 2 µL at 10 µM (separate tube)

5. Single band on gel: No contaminating products

Cleanup Methods

ExoSAP-IT (enzymatic cleanup): Column cleanup (Zymo DNA Clean & Concentrator): Gel extraction (Qiagen QIAquick): For ENTM201L COI sequencing:

Submission Checklist

Before submitting to sequencing core:

Troubleshooting Guide

Problem: Gel doesn't solidify

Causes: Solutions:

Problem: Wells collapse when loading

Causes: Solutions:

Problem: Samples leak into adjacent wells

Causes: Solutions:

Problem: Bands are curved ("smiling" or "frowning")

Causes: Solutions:

Problem: No fluorescence visible

Causes: Solutions:

Problem: Bands are streaky/comet-shaped

Causes: Solutions:

Safety Considerations

SYBR Safe Handling

Classification: Non-hazardous (not mutagenic or carcinogenic) Precautions: Spills:

UV Exposure

Hazards: Protection: For DNA recovery: Use blue light, not UV, to avoid damaging DNA before extraction

Electrical Safety

Hazards: Safety rules:

Real-World Applications in Mosquito Research

Species Identification Through DNA Barcoding

Workflow:

1. Extract DNA from unknown mosquito

2. PCR amplify COI barcode region (712 bp)

3. Gel electrophoresis confirms amplification

4. Sequence PCR product

5. BLAST against BOLD database

6. Species ID based on >97% identity

Gel's role: Quality control checkpoint before expensive sequencing Example: California Department of Public Health identifies 50+ mosquito species using COI barcoding. Gel electrophoresis is run on every sample to verify amplification before sequencing.

Pathogen Detection in Vector Surveillance

PCR-based detection of West Nile virus, Zika, dengue, malaria: Workflow:

1. Extract RNA from mosquito pools (25-50 mosquitoes)

2. Reverse transcription (RNA → cDNA)

3. PCR amplify viral genes (if present)

4. Gel shows band = pool is positive for pathogen

5. Quantification by qPCR (if needed)

Gel's role: Rapid screening Example: During West Nile virus outbreaks, mosquito control districts screen thousands of pools per month. Gel electrophoresis provides same-day results for public health response.

Insecticide Resistance Genotyping

kdr mutations in voltage-gated sodium channel confer pyrethroid resistance. PCR-RFLP method:

1. PCR amplify region containing kdr locus (~400 bp)

2. Digest with restriction enzyme (cuts wild-type, not mutant)

3. Gel shows fragment pattern:

- Wild-type: 400 bp cut → 250 bp + 150 bp

- Mutant: 400 bp uncut (enzyme site abolished)

- Heterozygote: All three bands (400, 250, 150 bp)

Gel's role: Genotype visualization Example: Studies of Aedes aegypti pyrethroid resistance in Florida use gel-based kdr genotyping to track resistance allele frequency over time.

Phylogenetic Studies and Population Genetics

Mitochondrial haplotype analysis: Workflow:

1. PCR amplify COI from multiple populations

2. Gel confirms all samples amplified successfully

3. Sequence all products

4. Align sequences, build phylogenetic tree

5. Infer population structure and migration patterns

Gel's role: Sample quality screening Example: Tracking invasive Aedes albopictus spread across USA using COI phylogeography. Gel electrophoresis screens 200+ samples before submitting best-quality products for sequencing.

Literature and Further Reading

Classic Papers on Electrophoresis

1. Principles of Agarose Gel Electrophoresis:

- Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press.

- Lee, P. Y., et al. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments 62: e3923. https://doi.org/10.3791/3923

2. DNA Staining Methods:

- Tuma, R. S., et al. (1999). Characterization of SYBR Gold nucleic acid gel stain: A dye optimized for use with 300-nm ultraviolet transilluminators. Analytical Biochemistry 268(2): 278-288. https://doi.org/10.1006/abio.1998.3067

- Schneeberger, C., et al. (1995). Quantitative detection of hepatitis B virus DNA in serum using a new rapid fluorescence-based PCR detection system. Journal of Clinical Microbiology 33(8): 2210-2216.

Mosquito DNA Barcoding Applications

3. COI Barcoding Protocols:

- Hebert, P. D. N., et al. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B 270(1512): 313-321. https://doi.org/10.1098/rspb.2002.2218

- Chan, A., et al. (2014). DNA barcoding: Complementing morphological identification of mosquito species in Singapore. Parasites & Vectors 7: 569. https://doi.org/10.1186/s13071-014-0569-4

4. Gel-Based Quality Control:

- Belgrader, P., et al. (1999). PCR detection of bacteria in seven minutes. Science 284(5413): 449-450. https://doi.org/10.1126/science.284.5413.449

- Lorenz, T. C. (2012). Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments 63: e3998. https://doi.org/10.3791/3998

Vector Surveillance and Pathogen Detection

5. Gel-Based Diagnostics:

- Crabtree, M. B., et al. (2003). Evaluation of a DNA vaccine against West Nile virus in an immunocompromised host. Vaccine 21(15): 1588-1595.

- Johnson, B. W., et al. (2005). Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 43(9): 4332-4338.

6. Insecticide Resistance Genotyping:

- Kasai, S., et al. (2014). PCR-based identification of pyrethroid-resistant kdr-type Aedes aegypti in Thailand. Pesticide Biochemistry and Physiology 116: 10-16. https://doi.org/10.1016/j.pestbp.2014.09.004

- Saavedra-Rodriguez, K., et al. (2007). A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Molecular Biology 16(6): 785-798.

Safety and Best Practices

7. SYBR Safe Safety Data:

- Invitrogen/Thermo Fisher Scientific. (2023). SYBR Safe DNA Gel Stain Safety Data Sheet.

- Singer, V. L., et al. (1999). Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantification. Analytical Biochemistry 249(2): 228-238.

8. Electrophoresis Troubleshooting:

- Voytas, D. (2000). Agarose gel electrophoresis. Current Protocols in Molecular Biology 51(1): 2.5A.1-2.5A.9. https://doi.org/10.1002/0471142727.mb0205as51

- Magdeldin, S., & Moser, A. (2012). Affinity Chromatography: Principles and Applications. InTech.

Key Takeaways

Gel Electrophoresis is Foundation of Molecular Biology

Despite being 50+ years old, gel electrophoresis remains essential because:

Understanding Physics Enables Troubleshooting

When gels fail, think about the physics:

Quality Control Checkpoint

Gel electrophoresis is the gatekeeper for downstream applications:

Never skip the gel. Sequencing a failed PCR wastes $15-30 per sample.

Real-World Impact

Every DNA-based diagnostic, forensic analysis, pathogen detection, and species identification workflow uses gel electrophoresis as quality control. Understanding this technique connects you to:

Connection to Lab Activities

In lab, you will:

1. Prepare 1% agarose gel with SYBR Safe staining

2. Load COI PCR products from alongside 1 kb ladder

3. Run electrophoresis at 100 V for 45-60 minutes

4. Visualize bands using blue light transilluminator

5. Interpret results:

- Successful PCR: Single band at 712 bp

- Failed PCR: No band (troubleshoot)

- Non-specific: Multiple bands (optimize)

6. Quantify by comparison to ladder bands

7. Decide on cleanup method (ExoSAP, column, or gel extraction)

8. Prepare samples for Sanger sequencing ()

Remember: The gel tells a story about your PCR. Learn to read it critically, and use that information to troubleshoot problems before wasting time and money on sequencing.
Document prepared for ENTM201L - General Entomology Laboratory UC Riverside, Department of Entomology Fall 2025

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