ENTM201L - General Entomology Laboratory | UC Riverside
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You have successfully extracted DNA from mosquito tissue. You have quantified it with Qubit and assessed purity with NanoDrop. Now comes the critical question: Is this DNA good enough for my downstream applications?
Not all DNA is created equal. DNA with the "correct" concentration might still fail in PCR if it is contaminated with inhibitors. DNA with perfect purity ratios might still produce poor sequencing results if it is fragmented. Understanding the three pillars of DNA quality - concentration, purity, and integrity - allows you to predict success, troubleshoot failures, and optimize your experimental workflow.
The PCR enzyme we use in ENTM201L has specific optimal conditions:
Template DNA Amount:The target gene (COI) exists as single copy per mitochondrial genome. Mosquito cells contain hundreds to thousands of mitochondria, so each cell has hundreds of COI copies. But the nuclear genome is huge (200 million base pairs), meaning statistically, at low DNA amounts, primers might not find target.
Calculation:Mosquito genome size: 200 Mb = 200,000,000 bp
Average genome mass: 200 Mb × 650 Da/bp × 1.67×10⁻²⁴ g/Da = 217 femtograms
Number of genomes in 25 ng DNA: 25 ng ÷ 217 fg = 115,000 genomes
Number of COI targets (at 500 mitochondria/cell): 115,000 × 500 = 57,500,000 copies
This is enough for robust PCR. Below 10 ng, you risk having too few target molecules for efficient exponential amplification.
| Inhibitor | Source | Mechanism | Solution |
|---|---|---|---|
| Proteins | Incomplete Proteinase K digestion | Bind polymerase or DNA | Dilute template 1:2; add BSA |
| Salts (NaCl, guanidine) | Lysis/binding buffers | Disrupt Mg²⁺ cofactor binding | Dilute template 1:5; extra washes |
| EDTA | Elution buffer (if present) | Chelates Mg²⁺, essential cofactor | Use Tris-only elution buffer |
| Ethanol | Incomplete drying | Denatures polymerase | Dry beads completely; do not over-dry |
| Melanin | Mosquito eye pigments | Absorbs UV, minor PCR inhibition | Dilute template 1:2 |
| Humic acids | Soil-preserved specimens | Bind polymerase | Use polymerase with BSA included |
| Heme | Blood-fed mosquitoes | Oxidative damage to dNTPs | Extract before blood meal digestion |
In Lab, your class is comparing two DNA extraction methods. Understanding their strengths and weaknesses helps you interpret results.
| Feature | Magnetic Beads (BioDynami) | Column (Zymo) |
|---|---|---|
| Principle | DNA binds beads in high salt; release in low salt | DNA binds silica membrane; elute in low salt |
| Mechanical stress | Minimal (no centrifugation) | High (14,000×g spin through membrane) |
| Expected fragment size | 50-150 kb | 10-50 kb |
| Yield from 1 mosquito | 20-60 ng/µL (in 50 µL) | 15-40 ng/µL (in 50 µL) |
| A260/A280 ratio | 1.80-1.95 | 1.75-1.90 |
| A260/A230 ratio | 1.9-2.2 | 1.8-2.0 |
| Time required | 75 min | 90 min |
| Cost per sample | ~$3 | ~$5 |
| Hands-on steps | Many (8+ pipetting steps) | Fewer (5-6 pipetting steps) |
| Automation potential | Easy (magnetic robots) | Difficult (centrifuge integration) |
| Best for | Long-read sequencing, genome assembly | Standard PCR, Sanger sequencing |
Expected yields vary by preservation method:
| Preservation | Magnetic Beads (ng/µL) | Column (ng/µL) | Difference |
|---|---|---|---|
| -80°C Frozen | 40-60 | 30-50 | Beads ~20% higher |
| 95% Ethanol | 25-45 | 20-35 | Beads ~15% higher |
| Silica Gel | 15-35 | 10-25 | Beads ~30% higher |
| 70% Ethanol | 8-20 | 5-15 | Beads ~40% higher |
1. Can you concentrate the sample?
- Yes → SpeedVac concentration (remove water by vacuum)
- Concentrate 50 µL to 15 µL = 3× concentration = 9 ng/µL
- Risk: Concentrates inhibitors too
- Yes → DNA precipitation
- Add sodium acetate + ethanol, pellet, resuspend in smaller volume
- Removes some salts during wash
- No → Proceed with more template volume (see below)
2. Can you use more template volume?
- Standard PCR: 1 µL template in 25 µL reaction
- Low DNA: Use 3-5 µL template in 25 µL reaction
- At 3 ng/µL, 5 µL provides 15 ng total (acceptable)
- Trade-off: More volume means more potential inhibitors
3. Can you increase PCR cycles?
- Standard: 30-35 cycles
- Low template: 35-40 cycles
- Each extra cycle doubles product (in theory)
- Trade-off: More cycles = more non-specific amplification
4. Should you re-extract?
- If A260/A280 and A260/A230 are good → Try PCR first
- If purity ratios are poor → Re-extract recommended
- If DNA <1 ng/µL → Definitely re-extract
Decision Path:
├─ Is A260/A280 >1.7?
│ ├─ YES → Acceptable for mosquito DNA, proceed to PCR
│ └─ NO → High protein contamination
│ ├─ Can you dilute? (If DNA conc >50 ng/µL)
│ │ └─ Dilute 1:2 with elution buffer
│ ├─ Can you add cleanup step?
│ │ └─ Zymo DNA Clean & Concentrator (removes proteins)
│ └─ Should you re-extract?
│ └─ Use more Proteinase K, longer incubation
Decision Path:
├─ Is A260/A230 >1.8?
│ ├─ YES → Acceptable, proceed to PCR
│ └─ NO → High salt contamination
│ ├─ Can you dilute? (If DNA conc >50 ng/µL)
│ │ └─ Dilute 1:5 (dilutes salts more than proteins)
│ ├─ Can you add extra wash?
│ │ └─ Add 80% ethanol, vortex, magnetic separation, dry
│ └─ Should you precipitate?
│ └─ Ethanol precipitation removes salts effectively
C₁V₁ = C₂V₂
Where:
C₁ = Initial concentration
V₁ = Volume of stock to use
C₂ = Desired final concentration
V₂ = Desired final volume
You have DNA at 80 ng/µL. You want 50 µL at 20 ng/µL for PCR setup.
C₁ = 80 ng/µL
V₁ = ? (what we're solving for)
C₂ = 20 ng/µL
V₂ = 50 µL
80 × V₁ = 20 × 50
80 × V₁ = 1000
V₁ = 1000 ÷ 80 = 12.5 µL
Mix: 12.5 µL DNA stock + 37.5 µL elution buffer = 50 µL at 20 ng/µL
You have DNA at 45 ng/µL. You want to make 100 µL at 10 ng/µL.
C₁ = 45 ng/µL
V₁ = ?
C₂ = 10 ng/µL
V₂ = 100 µL
45 × V₁ = 10 × 100
45 × V₁ = 1000
V₁ = 1000 ÷ 45 = 22.2 µL
Mix: 22.2 µL DNA stock + 77.8 µL elution buffer = 100 µL at 10 ng/µL
You have DNA at 5 ng/µL. You need 25 ng total in your PCR reaction. How much volume?
Needed: 25 ng
Concentration: 5 ng/µL
Volume = Amount ÷ Concentration = 25 ng ÷ 5 ng/µL = 5 µL
Add 5 µL of your DNA to PCR reaction (instead of standard 1 µL)
You have DNA at 200 ng/µL. You want to make 20 ng/µL (final) but you also want to save a 50 ng/µL intermediate stock.
Step 1: Make 50 ng/µL intermediate200 ng/µL × V₁ = 50 ng/µL × 100 µL
V₁ = 25 µL DNA + 75 µL buffer = 100 µL at 50 ng/µL
50 ng/µL × V₁ = 20 ng/µL × 50 µL
V₁ = 20 µL intermediate + 30 µL buffer = 50 µL at 20 ng/µL
After successful PCR in Lab, you will prepare your COI amplicons for Sanger sequencing. The sequencing facility has strict requirements.
Ladder Sample
| |
10 kb --- ===== ← Sharp band >20 kb (HMW genomic DNA)
5 kb ---
3 kb ---
1 kb ---
500 bp ---
Interpretation: High molecular weight DNA, intact
Ladder Sample
| |
10 kb ---::::
5 kb ---:::: ← Smear from 10 kb down to <1 kb
3 kb ---::::
1 kb ---::::
500 bp ---::
Interpretation: DNases active, DNA fragmented
Ladder Sample
| |
1 kb ---
750 bp --- ===== ← Single sharp band at 712 bp (COI)
500 bp ---
250 bp ---
Interpretation: Specific amplification, ready for sequencing
Ladder Sample
| |
1 kb ---
750 bp ---
500 bp --- (nothing visible)
250 bp ---
Interpretation: No amplification - check template quality/quantity
Ladder Sample
| |
1 kb ---
750 bp ---
500 bp ---
250 bp ---
100 bp ---::::: ← Smear/band at 50-100 bp
50 bp ---
Interpretation: Primers annealing to each other, not template
Ladder Sample
| |
1 kb --- ===
750 bp --- ===== ← Multiple bands (including correct size)
500 bp --- ===
250 bp --- ==
Interpretation: Primers annealing to wrong targets; lower annealing temp
In your lab report, you will compare magnetic bead extraction (BioDynami) with column extraction (Zymo). Here is what to analyze:
For each sample:
1. Qubit concentration (ng/µL)
2. NanoDrop concentration (ng/µL)
3. A260/A280 ratio
4. A260/A230 ratio
5. Total yield (concentration × volume)
6. PCR success (yes/no, band intensity)
7. Sequencing quality (Phred score, read length)
Null hypothesis: Mean concentration (beads) = Mean concentration (columns)
Alternative: Mean concentration (beads) ≠ Mean concentration (columns)
Significance level: α = 0.05
DNA quality is not just one number. It is the combination of:
All three must be adequate for your application.
When experiments fail:
1. Measure all three quality parameters
2. Identify which parameter is out of spec
3. Understand the molecular cause
4. Apply targeted solution
Do not blindly re-extract. Understand what went wrong and fix that specific problem.
Magnetic beads and columns both work, but excel at different applications:
Choose the method that matches your downstream application.
In lab, you will:
1. Measure DNA quality comprehensively
- Qubit for concentration
- NanoDrop for purity ratios
- Gel for integrity (if time permits)
2. Make decisions based on quality metrics
- Dilute if too concentrated
- Adjust template volume if too dilute
- Troubleshoot based on purity ratios
3. Compare extraction methods
- Analyze team data (beads vs. columns)
- Statistical comparison of yield and purity
- Discuss which method is "better" for your application
4. Optimize PCR based on DNA quality
- Use Qubit concentration for template calculations
- Add BSA if protein contamination suspected
- Dilute if inhibitors suspected
Remember: Quality control is not a checkbox. It is a diagnostic tool that tells you whether to proceed, optimize, or troubleshoot.This module is supported by peer-reviewed scientific literature on DNA quality assessment, purity metrics, and optimization strategies for molecular biology applications.
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