ENTM201L - General Entomology Laboratory | UC Riverside
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While Qubit fluorometry tells us the concentration of double-stranded DNA with high accuracy, it provides no information about sample purity. Is your DNA contaminated with proteins from incomplete lysis? Are salts from extraction buffers still present? Did RNA survive despite RNase treatment? These questions are critical because contaminants can inhibit downstream applications like PCR and sequencing.
NanoDrop spectrophotometry answers these questions by measuring how much ultraviolet and visible light your sample absorbs across multiple wavelengths. Different molecules absorb light at different wavelengths, creating a unique "fingerprint" that reveals both what is present and how pure your DNA is.
Think of it this way: Qubit tells you "how much DNA," while NanoDrop tells you "how pure that DNA is."
When light passes through a solution, some wavelengths are absorbed by molecules in that solution. The amount of absorption depends on:
1. Molecular structure - Specific chemical bonds absorb specific wavelengths
2. Concentration - More molecules mean more absorption
3. Path length - Light traveling through more solution gets absorbed more
Absorbance (A) is defined as:A = -log₁₀(I/I₀)Where:
DNA and RNA strongly absorb ultraviolet light because of their chemical structure. Specifically:
Purine and pyrimidine bases (A, T, G, C, U) contain aromatic rings - rings of carbon atoms with delocalized electrons. These electrons can absorb UV photons and jump to higher energy states (called π → π* transitions). Maximum absorption occurs at 260 nm for all nucleic acids because this wavelength matches the energy difference between ground and excited electronic states in the aromatic bases.Different molecules have different absorption maxima:
By measuring absorbance at these three wavelengths (260, 280, 230 nm), we can determine:
1. Nucleic acid concentration from A260
2. Protein contamination from A260/A280 ratio
3. Salt/organic contamination from A260/A230 ratio
The relationship between absorbance, concentration, and path length is described by the Beer-Lambert Law:
A = ε × c × lWhere:
DNA concentration (ng/µL) = A260 × 50 × dilution factorThis calculation only works if your sample contains pure dsDNA with no contaminants. If proteins, RNA, or other UV-absorbing molecules are present, the A260 reading will be inflated, giving falsely high concentration estimates.
This is why we calculate purity ratios - to assess whether our sample meets the "pure DNA" assumption.
Traditional spectrophotometers require cuvettes - small rectangular containers that hold your sample in a defined path length (usually 1 cm). You need hundreds of microliters to fill a cuvette, which wastes precious DNA samples.
NanoDrop revolutionized this with a micro-volume platform that requires only 1-2 µL.1. Lower pedestal - You pipette 1-2 µL onto this surface
2. Upper pedestal - Lowered onto the sample droplet
3. Surface tension - Liquid spreads between pedestals, creating a defined column
4. Path length - Adjustable from 0.05 mm (1 mm for NanoDrop One) to 1 mm
- This allows measurement of concentrated samples without dilution
Light path:Xenon flash lamp → Wavelength selector → Sample column → Detector array| Feature | NanoDrop | Traditional Spectrophotometer |
|---|---|---|
| Sample volume | 1-2 µL | 50-500 µL (cuvette) |
| Sample recovery | 100% (pipette back) | Difficult (trapped in cuvette) |
| Speed | 3-5 seconds | 30-60 seconds |
| Cleaning | Wipe with Kimwipe | Extensive washing required |
| Dynamic range | 0.04-15,000 ng/µL (via path length adjustment) | 0.04-200 ng/µL (single path) |
| Cost per measurement | Negligible | Requires cuvettes |
Pure DNA has an A260/A280 ratio of 1.8-2.0. This ratio tells us whether proteins are contaminating our sample.
Why 280 nm for proteins?- Tryptophan (Trp): Strong absorbance at 280 nm
- Tyrosine (Tyr): Moderate absorbance at 280 nm
- Phenylalanine (Phe): Weak absorbance at 260 nm
Most proteins contain these amino acids and therefore absorb at 280 nm.
| A260/A280 Ratio | Interpretation | Likely Cause | Action Required |
|---|---|---|---|
| 1.8-2.0 | Pure DNA | Successful extraction | None - proceed with downstream applications |
| <1.8 | Protein contamination | Incomplete Proteinase K digestion | Add more Proteinase K; extend incubation; repeat wash steps |
| >2.0 | RNA contamination or very pure DNA | No RNase treatment or excellent extraction | If RNA suspected: add RNase; if pure DNA: this is acceptable |
| <1.5 | Heavy protein contamination | Failed lysis or insufficient washing | Repeat extraction with longer lysis time |
| >2.2 | Significant RNA or unusual contamination | RNA or phenol carryover | Add RNase treatment or organic extraction cleanup |
This indicates protein contamination. Here's why:
When proteins are present:
Pure DNA:
A260 = 0.20 (from DNA)
A280 = 0.10 (from DNA)
Ratio = 0.20/0.10 = 2.0
With protein contamination:
A260 = 0.20 (from DNA) + 0.02 (from protein) = 0.22
A280 = 0.10 (from DNA) + 0.08 (from protein) = 0.18
Ratio = 0.22/0.18 = 1.22
Many extraction reagents and contaminants absorb at 230 nm:
Pure DNA has minimal absorbance at 230 nm, so the A260/A230 ratio should be 2.0-2.2 (some sources say 2.2-2.5).
| A260/A230 Ratio | Interpretation | Likely Cause | Action Required |
|---|---|---|---|
| 2.0-2.2 | Pure DNA, minimal salt | Successful washing | None - proceed |
| 1.5-2.0 | Moderate contamination | Residual binding buffer or incomplete washing | Add extra ethanol wash step |
| <1.5 | Heavy salt contamination | Ethanol not fully removed or binding buffer carryover | Repeat extraction with careful washing |
| >2.2 | Very pure or degraded DNA | Excellent technique or DNA fragmentation | Check on gel for integrity |
| <1.0 | Severe contamination | Major protocol error | Repeat extraction from beginning |
This indicates salt or organic contamination:
Pure DNA:
A260 = 0.20 (from DNA)
A230 = 0.09 (from DNA background)
Ratio = 0.20/0.09 = 2.22
With guanidine salt contamination:
A260 = 0.20 (from DNA) + 0.01 (from salt) = 0.21
A230 = 0.09 (from DNA) + 0.06 (from salt) = 0.15
Ratio = 0.21/0.15 = 1.4
1. Check A260/A280 ratio - if <1.8, protein contamination
2. Check A260/A230 ratio - if <2.0, salt/organic contamination
3. Run gel electrophoresis - if smear instead of band, degradation
4. Consider RNA - if no RNase used, RNA inflates A260
Trust Qubit for PCR setup, but use NanoDrop ratios for troubleshooting.| Preservation | Expected Qubit (ng/µL) | Expected A260/A280 | Expected A260/A230 | Notes |
|---|---|---|---|---|
| -80°C Frozen | 30-60 | 1.85-1.95 | 2.0-2.2 | Best quality; highest yield |
| 95% Ethanol | 20-40 | 1.80-1.90 | 1.9-2.1 | Good quality; ethanol residue may lower A230 |
| Silica Gel | 10-30 | 1.75-1.90 | 1.8-2.0 | Variable; depends on drying speed |
| 70% Ethanol | 5-15 | 1.70-1.85 | 1.7-2.0 | Degraded; low ratios common |
1. Accuracy is critical
- Setting up PCR reactions
- Normalizing DNA for sequencing libraries
- Quantifying low-concentration samples (<10 ng/µL)
2. RNA contamination is suspected
- No RNase treatment was used
- Need dsDNA concentration specifically
3. Contaminants are present
- Melanin from insect eyes/cuticle
- Residual phenol from extractions
- High salt concentrations
4. Small sample volumes
- Only 10 µL total DNA extracted
- Cannot spare sample for multiple measurements
1. Purity assessment needed
- Troubleshooting failed PCR
- Checking effectiveness of cleanup steps
- Verifying protein digestion
2. Quick screening
- Many samples to process
- Just need rough concentration estimate
- Reagent cost is a concern (Qubit uses consumables)
3. High concentration samples
- >100 ng/µL (above Qubit range without dilution)
- Plasmid DNA or PCR products
4. Full spectrum data wanted
- Unusual contaminants suspected
- Research-grade characterization
- Publication-quality documentation
1. NanoDrop first (fast, no reagents)
- Check A260/A280 and A260/A230 ratios
- Get rough concentration estimate
- Identify gross contamination
2. Qubit second (accurate dsDNA quantification)
- Get precise dsDNA concentration
- Use for PCR calculations
- Compare to NanoDrop to identify specific contaminants
Interpretation:1. Increase Proteinase K concentration
- Use 2× recommended amount
- Or add second aliquot after 30 min
2. Extend lysis time
- 60-90 min instead of 45 min
- Check for "turbid to clear" transition
3. Raise temperature
- 58-60°C instead of 56°C (still below DNA denaturation)
- Accelerates protease activity
4. Add extra wash step
- Third ethanol wash removes protein debris
5. Use cleanup column
- Zymo DNA Clean & Concentrator
- Removes proteins while retaining DNA
For PCR despite low ratio:1. Improve washing technique
- Ensure complete ethanol removal between washes
- Use fresh 80% ethanol (not reused)
- Pulse spin after final wash to collect residual ethanol
2. Dry completely but not excessively
- 1-2 min air dry with tube open
- Do not over-dry (>5 min) or beads crack
3. Add extra ethanol wash
- Third or fourth wash removes stubborn salts
- Use 100% ethanol for final wash
4. Dilute and re-precipitate
- Dilute DNA 1:10 in TE buffer
- Add 0.1× volume 3M sodium acetate pH 5.2
- Add 2.5× volume cold 100% ethanol
- Incubate -20°C for 30 min
- Centrifuge 15 min at 14,000×g
- Wash pellet with 70% ethanol
- Resuspend in fresh elution buffer
For PCR despite low ratio:Crime scene samples often have:
Museum specimens (>50 years old) present challenges:
Circulating tumor DNA (ctDNA) in blood:
1. Spectrophotometry Principles:
- Wilfinger, W. W., et al. (1997). Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22(3): 474-481. https://doi.org/10.2144/97223st01
- Manchester, K. L. (1995). Value of A260/A280 ratios for measurement of purity of nucleic acids. BioTechniques 19(2): 208-210.
2. NanoDrop Technology:
- Desjardins, P., & Conklin, D. (2010). NanoDrop microvolume quantitation of nucleic acids. Journal of Visualized Experiments 45: e2565. https://doi.org/10.3791/2565
- Gallagher, S. R., & Desjardins, P. R. (2011). Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current Protocols in Molecular Biology 93: A.3D.1-A.3D.14. https://doi.org/10.1002/0471142727.mba03ds93
3. Purity Assessment and Troubleshooting:
- Glasel, J. A. (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques 18(1): 62-63.
- Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: The past and the present. Journal of Biomedicine and Biotechnology 2009: 574398. https://doi.org/10.1155/2009/574398
4. Contaminant Effects on PCR:
- Schrader, C., et al. (2012). PCR inhibitors - occurrence, properties and removal. Journal of Applied Microbiology 113(5): 1014-1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x
- Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology 63(10): 3741-3751. https://doi.org/10.1128/aem.63.10.3741-3751.1997
5. Mosquito DNA Quality:
- Lawrence, A. L., et al. (2019). Comparison of five DNA extraction methods for DNA barcoding of mosquitoes. Journal of Medical Entomology 56(4): 1148-1153. https://doi.org/10.1093/jme/tjz036
- Ballinger-Crabtree, M. E., et al. (1992). Comparison of extraction techniques for mosquito DNA. Journal of the American Mosquito Control Association 8(3): 314-316.
6. Beer-Lambert Law Applications:
- Swinehart, D. F. (1962). The Beer-Lambert Law. Journal of Chemical Education 39(7): 333-335. https://doi.org/10.1021/ed039p333
7. Comparison of Quantification Methods:
- Simbolo, M., et al. (2013). DNA qualification workflow for next generation sequencing of histopathological samples. PLoS ONE 8(6): e62692. https://doi.org/10.1371/journal.pone.0062692
These instruments are not competitors - they provide different, complementary information:
Qubit = Highly accurate measurement of how much dsDNA NanoDrop = Assessment of how pure that DNA isTogether, they give you the complete picture needed for informed decisions about downstream applications.
When your PCR fails, purity ratios tell you why:
Insect DNA extractions commonly show:
In lab, you will:
1. Measure DNA on both NanoDrop and Qubit
- Compare concentrations between methods
- Assess purity ratios
- Identify which samples need cleanup
2. Make decisions for PCR
- Calculate template volumes based on Qubit
- Dilute if necessary
- Add BSA to reactions with low A260/A280
3. Interpret discrepancies
- If NanoDrop >> Qubit → Identify likely contaminant
- Use ratios to guide troubleshooting
4. Compare preservation methods
- Do frozen samples have better ratios?
- Does 70% ethanol show salt contamination?
Remember: These measurements are not just quality control checkboxes. They are diagnostic tools that tell you about the molecular composition of your sample and guide your experimental strategy.View comprehensive literature references and citations supporting this module's content.
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