ENTM201L - General Entomology Laboratory | UC Riverside |
This protocol guides you through comprehensive assessment of DNA quality using three complementary metrics: concentration (Qubit), purity (NanoDrop), and integrity (gel electrophoresis). Understanding all three metrics allows you to predict PCR success and troubleshoot failures.
Always vortex DNA samples before pipetting to ensure homogeneous distribution. Settled DNA will give inaccurate readings.
1 Calculate reagent volumes needed:
Number of samples + 2 standards + 1 extra = Total tubes
Example: 8 samples + 2 standards + 1 = 11 tubes
Working solution per tube: 199 µL
Total working solution: 11 × 199 = 2,189 µL (round to 2,200 µL)2 Mix Qubit reagent and buffer:
Prepare working solution fresh. Dye can photobleach if exposed to light for >30 minutes. If preparing in advance, wrap tube in foil.
3 Label two Qubit assay tubes "S1" (0 ng/µL) and "S2" (100 ng/µL)
4 Add to each standard tube:
5 For each DNA sample:
1 µL sample + 199 µL working solution = 200× dilution
If Qubit reads 0.25 ng/µL, your original concentration is: 0.25 × 200 = 50 ng/µL
6 Incubate all tubes (standards + samples) at room temperature for 2 minutes
7 On Qubit Flex:
"Too low" reading: DNA concentration below detection limit (0.005 ng/µL). Use 2 µL sample instead of 1 µL and divide result by 2.
"Too high" reading: DNA saturating detector. Dilute sample 1:10 with elution buffer and re-measure.
1 Clean NanoDrop pedestals:
2 Measure blank (elution buffer used for DNA extraction):
Always use the same elution buffer that your DNA is in. TE buffer and water have different absorbance properties.
3 For each sample:
| Metric | Pure DNA | Acceptable Range | Interpretation if Low |
|---|---|---|---|
| A260/A280 | 1.8-2.0 | 1.75-1.90 (mosquito DNA) | Protein contamination; incomplete Proteinase K digestion |
| A260/A230 | 2.0-2.2 | 1.8-2.0 (acceptable) | Salt contamination; guanidine carryover; incomplete ethanol removal |
A260/A280 ratios of 1.75-1.85 are normal for mosquito DNA due to sclerotized cuticle proteins that resist Proteinase K. These ratios are still PCR-compatible.
| Observation | Likely Cause | Action |
|---|---|---|
| NanoDrop >> Qubit (30-50% higher) | RNA contamination or melanin absorbance | Trust Qubit for PCR calculations |
| NanoDrop ≈ Qubit, good ratios | Pure DNA, minimal contamination | Proceed with confidence to PCR |
| NanoDrop high, Qubit low, A260/A280 < 1.7 | Heavy protein contamination | Dilute 1:2 for PCR; add BSA to reaction |
| NanoDrop high, Qubit low, A260/A230 < 1.5 | Salt contamination | Dilute 1:5 for PCR or re-extract with extra washes |
For PCR setup: Always use Qubit concentration to calculate template volume. Use NanoDrop ratios for troubleshooting if PCR fails.
Problem: Qubit reads 85 ng/µL. Optimal PCR range is 10-50 ng/µL.
Goal: Make 30 µL at 20 ng/µL
C₁V₁ = C₂V₂
85 × V₁ = 20 × 30
85 × V₁ = 600
V₁ = 7.1 µL
Mix: 7.1 µL DNA stock + 22.9 µL elution buffer = 30 µL at 20 ng/µLProblem: Qubit reads 4 ng/µL. Need 25 ng total for PCR.
Solution: Use more template volume
Volume = Amount needed ÷ Concentration
Volume = 25 ng ÷ 4 ng/µL = 6.25 µL
Use 6.25 µL (or round to 6 µL) in PCR instead of standard 1 µL| Sample ID | Qubit (ng/µL) | NanoDrop (ng/µL) | A260/A280 | A260/A230 | Quality Assessment |
|---|---|---|---|---|---|
| Sample 1 | _______ | _______ | _______ | _______ | _______ |
| Sample 2 | _______ | _______ | _______ | _______ | _______ |
| Sample 3 | _______ | _______ | _______ | _______ | _______ |
If purity ratios are poor but you proceed to PCR anyway: dilute template 1:5 and add BSA to PCR at 0.4 µg/µL final concentration. This dilutes inhibitors while maintaining adequate template.
The quality metrics measured today determine your PCR strategy:
By completing this protocol, you have learned to make informed, data-driven decisions about DNA quality and PCR optimization - essential skills for any molecular biology workflow.