Gel Electrophoresis Protocol
Module 08: DNA Quality Assessment and PCR Product Analysis
Overview
This protocol describes agarose gel electrophoresis for visualizing DNA quality from extraction methods and analyzing PCR product success. You will run three critical gels to assess genomic DNA integrity and COI amplification success.
Learning Objectives
- Prepare and cast agarose gels with appropriate percentage for DNA visualization
- Load DNA samples with loading dye correctly
- Run electrophoresis at proper voltage and duration
- Visualize DNA bands using UV transillumination
- Interpret band patterns for DNA quality and PCR success
- Compare extraction methods based on gel results
Safety Considerations
- GelRed Stain: Intercalating dye - wear gloves when handling gels
- UV Light: Wear protective face shield and never look directly at UV source
- Electrical Hazard: Never open electrophoresis chamber while power is on
- Hot Agarose: Molten agarose can cause severe burns - handle with care
- Disposal: GelRed-stained gels go in hazardous waste container
Materials and Equipment
Equipment
- Microwave or hot plate
- Gel casting tray with combs (8-well or 12-well)
- Horizontal electrophoresis apparatus
- Power supply (capable of 50-150V)
- UV transilluminator or blue light imaging system
- Gel documentation system (camera)
- Micropipettes (P2, P10, P20)
Reagents
- Agarose powder
- 1× TAE buffer (Tris-Acetate-EDTA, pH 8.0)
- GelRed nucleic acid stain (10,000× stock)
- 6× DNA loading dye (with bromophenol blue and glycerol)
- DNA ladder (100 bp or 1 kb ladder)
Samples
- Genomic DNA from column extraction ()
- Genomic DNA from magnetic bead extraction ()
- PCR products from COI amplification ()
Protocol
Part 1: Gel Preparation (20 minutes)
Gel 1 & 2: Genomic DNA Quality Check (0.8% Agarose)
Purpose: Visualize high molecular weight genomic DNA (10-40 kb)
- Calculate agarose needed:
- For 50 mL gel: 0.4 g agarose (0.8%)
- For 100 mL gel: 0.8 g agarose (0.8%)
- Prepare agarose solution:
- Add agarose powder to flask
- Add appropriate volume of 1× TAE buffer
- Swirl to mix
- Melt agarose:
- Microwave on high for 45-60 seconds
- Swirl carefully (hot!)
- Continue heating in 15-second intervals until completely dissolved
- Solution should be completely clear with no particles
- Cool and add GelRed:
- Allow solution to cool to ~60°C (comfortable to touch flask)
- Add 5 µL GelRed per 50 mL gel (10 µL per 100 mL)
- Swirl gently to mix (avoid bubbles)
- Cast gel:
- Place gel tray on level surface
- Insert comb (8-well or 12-well)
- Pour agarose slowly to avoid bubbles
- Remove any bubbles with pipette tip
- Allow to solidify for 15-20 minutes (gel becomes opaque)
Gel 3: PCR Products (1.5% Agarose)
Purpose: Resolve 710 bp COI amplicon and detect primer dimers
- Calculate agarose needed:
- For 50 mL gel: 0.75 g agarose (1.5%)
- For 100 mL gel: 1.5 g agarose (1.5%)
- Follow steps 2-5 above for melting and casting
Part 2: Sample Preparation (10 minutes)
Preparing DNA Samples
| Sample Type |
DNA Volume |
Loading Dye |
Total Volume |
| Genomic DNA |
5 µL |
1 µL (6×) |
6 µL |
| PCR Product |
5 µL |
1 µL (6×) |
6 µL |
| DNA Ladder |
5 µL |
1 µL (6×) |
6 µL |
Procedure:
- Label PCR strip tubes or microcentrifuge tubes for each sample
- Add 5 µL of DNA sample to each tube
- Add 1 µL of 6× loading dye
- Mix by pipetting up and down 3 times
- Spin briefly to collect sample at bottom
- Keep on ice until loading
Part 3: Gel Loading and Electrophoresis (40 minutes)
Loading the Gel
- Prepare electrophoresis chamber:
- Remove comb carefully from solidified gel
- Place gel in electrophoresis chamber
- Add 1× TAE buffer until gel is submerged (~2-3 mm above gel)
- Ensure wells are completely filled with buffer
- Load samples:
- Set pipette to 6 µL
- Lane 1: Load 5 µL DNA ladder
- Lanes 2-6: Load genomic DNA samples (5 preservation methods)
- Insert pipette tip into well at 45° angle
- Dispense slowly - sample should sink to bottom
- Avoid piercing bottom of well or disturbing adjacent wells
Sample Loading Order
Gel 1: Column DNA ()
| Lane |
Sample |
Preservation Method |
| 1 |
Ladder |
- |
| 2 |
Sample A |
-80°C freezer |
| 3 |
Sample B |
RNAlater-ICE |
| 4 |
Sample C |
95% ethanol |
| 5 |
Sample D |
Silica gel |
| 6 |
Sample E |
70% ethanol |
Gel 2: Magnetic Bead DNA ()
Same layout as Gel 1, using samples F-J
Gel 3: PCR Products
Same layout, using PCR products from each preservation method
Running Electrophoresis
- Connect power supply:
- Red electrode (positive) to red terminal - far end of chamber
- Black electrode (negative) to black terminal - well end
- DNA migrates toward positive electrode (red)
- Set voltage:
- Genomic DNA gels: 80-100V
- PCR product gels: 100-120V
- Start electrophoresis:
- Turn on power supply
- Verify bubbles form at electrodes (indicates current flowing)
- Watch loading dye move through gel
- Monitor progress:
- Bromophenol blue dye migrates at ~300-500 bp
- Run until dye reaches 2/3 to 3/4 down gel
- Typical runtime: 30-45 minutes
- Stop electrophoresis:
- Turn off power supply first
- Disconnect electrodes
- Carefully remove gel from chamber
Part 4: Gel Imaging and Analysis (15 minutes)
Visualizing DNA
- Transfer gel to transilluminator:
- Wear UV protective face shield
- Place gel on UV transilluminator or blue light platform
- Remove any bubbles or liquid on gel surface
- Image gel:
- Turn on UV light (312 nm) or blue light
- Adjust exposure settings for optimal image
- Capture image with gel documentation system
- Include ruler or scale bar if available
- Label image:
- Date, gel number, sample IDs
- Voltage and runtime
- Ladder used
Interpreting Results
High-Quality Genomic DNA:
- Single bright band or tight smear in high molecular weight region (>10 kb)
- Prominent band at ~16 kb (circular mitochondrial genome)
- No low molecular weight smearing (indicates degradation)
- No diffuse smear throughout lane (indicates RNA contamination)
Successful PCR:
- Single bright band at ~710 bp (COI amplicon)
- No bands in negative control
- No primer dimers (<100 bp)
- No non-specific bands at other sizes
Failed PCR:
- No band at expected size
- Smearing (over-amplification or degraded template)
- Multiple bands (non-specific amplification)
- Only primer dimers visible
Data Recording
Preservation Method Comparison Table
| Sample |
Preservation |
DNA Quality (1-5) |
PCR Success |
Band Intensity |
Notes |
| A/F |
-80°C |
___ |
Y/N |
___ |
|
| B/G |
RNAlater-ICE |
___ |
Y/N |
___ |
|
| C/H |
95% ethanol |
___ |
Y/N |
___ |
|
| D/I |
Silica gel |
___ |
Y/N |
___ |
|
| E/J |
70% ethanol |
___ |
Y/N |
___ |
|
Troubleshooting
| Problem |
Possible Cause |
Solution |
| No DNA bands visible |
Concentration too low |
Load more sample (up to 10 µL) |
| Smeared bands |
DNA degraded |
Extract fresh DNA, avoid freeze-thaw |
| Bands in wrong location |
Gel ran backwards |
Check electrode polarity |
| Distorted bands |
Too much DNA loaded |
Dilute sample and reload |
| Gel melted during run |
Voltage too high |
Reduce to 100V, check buffer level |
| No current flow |
Electrodes not connected |
Check connections, ensure buffer covers gel |
Expected Results by Preservation Method
Genomic DNA Quality
- -80°C freezer: Excellent - bright high MW band, minimal degradation
- RNAlater-ICE: Excellent - similar to -80°C, very high quality
- 95% ethanol: Good to moderate - some degradation possible
- Silica gel: Moderate - more degradation, lower MW smear
- 70% ethanol: Poor - significant degradation, low MW smear
PCR Success Rates
Expected correlation between DNA quality and PCR success:
- Excellent DNA quality → Bright PCR band
- Good DNA quality → Moderate PCR band
- Poor DNA quality → Weak or no PCR band
Post-Lab Analysis
Quantitative Analysis
- Band intensity: Use ImageJ to quantify band intensity
- Success rate: Calculate % PCR success per preservation method
- Statistical comparison: Compare column vs. bead extraction methods
Questions to Consider
- Which preservation method yielded highest quality DNA?
- Did extraction method (column vs. beads) affect DNA quality?
- Was DNA quality predictive of PCR success?
- Which samples should be selected for sequencing?
- What factors contributed to PCR failures?
Waste Disposal
- GelRed-stained gels: Hazardous waste container (intercalating dye)
- TAE buffer: Can be reused 3-5 times, then dispose down drain
- Pipette tips: Regular biohazard waste
- Gloves: Regular trash (if no hazardous contact)
Key Takeaways
- Gel electrophoresis separates DNA by size through an electric field
- Agarose percentage determines resolution range (0.8% for large DNA, 1.5% for PCR products)
- DNA quality directly impacts PCR success rates
- Preservation method significantly affects DNA integrity
- Multiple assessment methods (gel, spectrophotometry, PCR) provide comprehensive quality evaluation
- Proper gel documentation is essential for reproducibility and publication