ENTM201L - General Entomology Laboratory | UC Riverside |
This protocol uses UV-Vis spectrophotometry to measure DNA purity via A260/A280 and A260/A230 ratios. These ratios detect protein and salt contamination that could inhibit PCR. While NanoDrop provides concentration estimates, trust Qubit for accurate dsDNA quantification and use NanoDrop for purity troubleshooting.
NanoDrop uses a Xenon flash lamp that emits UV light. Never look directly at the measurement pedestal when the arm is lowered during measurement. UV exposure can damage eyes.
1 Turn on NanoDrop spectrophotometer (power button on rear or side)
2 Launch NanoDrop software on computer
3 Wait for instrument initialization (approximately 30 seconds)
4 Select "Nucleic Acid" module in software
If the instrument was used previously, pedestals may have dried sample residue. Always start with cleaning step even if pedestals look clean.
5 Pipette 2 µL deionized water onto lower pedestal
6 Lower sampling arm to spread liquid between pedestals
7 Raise arm, wipe both upper and lower pedestals with clean Kimwipe
8 Repeat cleaning cycle 2-3 times until no residue visible
Residue from previous samples will contaminate your readings. Clean thoroughly between sample sets. Dried guanidine salts (from extraction buffers) appear as white crystals - these must be removed.
The blank establishes baseline absorbance for your elution buffer. If your DNA is in TE buffer, blank with TE. If in water, blank with water. Buffer and sample must match or readings will be inaccurate.
1 Ensure pedestals are clean and dry
2 Pipette 1-2 µL elution buffer (TE or water) onto lower pedestal
3 Lower sampling arm gently to create liquid column
4 In software, select "Blank" button
5 Wait 3-5 seconds for measurement
6 Software will display "Blank successful" message
7 Raise arm, wipe pedestals with Kimwipe
Blank only once at the start of a measurement session. Do not re-blank between samples unless you change elution buffers or have a long gap (>30 min) between measurements.
1 Wipe pedestals clean with Kimwipe
2 Vortex DNA sample tube for 5 seconds (critical - DNA settles)
3 Quick spin tube to collect liquid at bottom
4 Pipette 1-2 µL DNA onto lower pedestal
Use a P2 pipette set to 1.5 µL for best accuracy. Avoid introducing bubbles. If bubble forms, wipe pedestal and re-pipette. Bubbles scatter light and give false readings.
5 Lower sampling arm to spread DNA between pedestals
6 In software, enter Sample ID (e.g., "Sample1_MagBeads")
7 Click "Measure" button
8 Wait 3-5 seconds for measurement
Software will display spectral curve (190-840 nm) and calculated values:
| Metric | What to Record | What It Means |
|---|---|---|
| Concentration | ng/µL (dsDNA) | Estimate of DNA amount (compare to Qubit) |
| A260 | Absorbance value | Raw absorbance at 260 nm (nucleic acids) |
| A260/A280 | Ratio (unitless) | Protein purity indicator (target: 1.8-2.0) |
| A260/A230 | Ratio (unitless) | Salt/organic purity indicator (target: 2.0-2.2) |
9 Record all four values in lab notebook
10 Optional: Click "Export" to save full spectrum as PDF
11 Raise sampling arm
12 Immediately pipette DNA sample back into original tube
NanoDrop is non-destructive. You can recover your 1-2 µL sample if needed. This is useful for low-yield extractions where every microliter counts. Recovery rate: ~80-90%.
13 Wipe both pedestals with Kimwipe
14 Repeat Steps 1-12 for each DNA sample
Always wipe pedestals between samples. Carryover of just 0.1 µL from a high-concentration sample can contaminate low-concentration readings.
| Ratio | Interpretation | Likely Cause | PCR Impact |
|---|---|---|---|
| 1.8-2.0 | Pure DNA | Successful extraction | Proceed with confidence |
| 1.75-1.8 | Slight protein contamination (acceptable for mosquito DNA) | Sclerotized cuticle proteins | PCR usually succeeds |
| 1.5-1.75 | Moderate protein contamination | Incomplete Proteinase K digestion | Dilute 1:2; add BSA to PCR |
| <1.5 | Heavy protein contamination | Lysis failure or insufficient washing | Re-extract recommended |
| >2.0 | RNA contamination or very pure DNA | No RNase treatment | Usually acceptable; RNA doesn't inhibit PCR much |
| Ratio | Interpretation | Likely Cause | Action Required |
|---|---|---|---|
| 2.0-2.2 | Pure DNA, minimal salts | Excellent washing | None - proceed to PCR |
| 1.8-2.0 | Acceptable purity | Trace salt carryover | Proceed to PCR (monitor for inhibition) |
| 1.5-1.8 | Moderate salt contamination | Incomplete ethanol removal or guanidine carryover | Dilute 1:5 for PCR |
| <1.5 | Heavy contamination | Binding buffer carryover; ethanol not evaporated | Re-extract with extra washes |
| >2.2 | Very pure or degraded DNA | Excellent technique or DNA fragmentation | Check integrity on gel |
Example: NanoDrop = 40 ng/µL, Qubit = 15 ng/µL
Interpretation:
Action: Trust Qubit for PCR calculations. NanoDrop is overestimating due to contaminants.
Example: NanoDrop = 22 ng/µL, Qubit = 20 ng/µL, A260/A280 = 1.88, A260/A230 = 2.05
Interpretation: Pure dsDNA with minimal contamination
Action: Proceed to PCR with confidence. Either concentration value is acceptable.
Example: NanoDrop = 18 ng/µL, A260/A280 = 1.65, A260/A230 = 1.40
Interpretation: Heavy protein AND salt contamination
Action:
Causes and Solutions:
Cause: Absorbance at 230 nm is higher than at 260 nm (severe contamination)
Interpretation: Massive guanidine salt carryover or phenol contamination
Action: Do not use for PCR. Re-extract DNA from tissue.
Cause: RNA contamination or buffer mismatch
Check: Did you blank with the correct elution buffer?
Action:
Cause: Blank was contaminated or sample is very dilute
Action:
1 Pipette 2 µL deionized water onto lower pedestal
2 Lower arm, raise arm, wipe both pedestals thoroughly
3 Repeat cleaning 2-3 times
4 Lower sampling arm onto clean pedestal (protects optics)
5 Close NanoDrop software
6 Power off instrument (optional - can be left on)
Always leave the sampling arm lowered when not in use. This protects the optical surfaces from dust and reduces photobleaching of the measurement spot.
| Sample ID | NanoDrop (ng/µL) | A260 | A260/A280 | A260/A230 | Quality Assessment |
|---|---|---|---|---|---|
| Blank (Buffer) | 0.0 | 0.000 | — | — | Baseline |
| Sample 1 | _______ | _______ | _______ | _______ | _______ |
| Sample 2 | _______ | _______ | _______ | _______ | _______ |
| Sample 3 | _______ | _______ | _______ | _______ | _______ |
Use NanoDrop and Qubit together. NanoDrop for purity troubleshooting, Qubit for accurate quantification. Together, they give you complete quality assessment for informed PCR decisions.