The nucleic acid protein analyzer is a key equipment used in molecular biology laboratories for rapid quantification of nucleic acid (DNA/RNA) and protein concentrations. Proper operation not only ensures the accuracy of data, but also extends the lifespan of the instrument. This article will introduce the basic operating specifications, common problems, and practical skills of nucleic acid protein analyzers for beginners in the laboratory.
1、 Preparation work before operation
1. Instrument calibration
-Blank calibration: Before each use, ultrapure water or buffer solution (such as TE buffer) should be used for blank calibration to eliminate background interference.
-Regular maintenance: If the instrument has not been used for a long time, it needs to be recalibrated and the light source and detector checked for normal operation.
2. Sample preparation
-Nucleic acid samples: Ensure that there are no particles or impurities, and if necessary, centrifuge (12000rpm, 1-2 minutes) to remove impurities.
-Protein samples: Avoid high concentration detergents (such as SDS) to prevent them from affecting absorbance detection.
3. Choose the appropriate detection mode
-UV absorption method (such as NanoDrop): suitable for rapid determination of DNA/RNA (260nm) and proteins (280nm).
-Fluorescence method (such as Qubit): suitable for low concentration nucleic acid detection, with higher sensitivity.
2、 Standard Operating Procedure
1. Startup and initialization
-Turn on the instrument and preheat for 5-10 minutes (some devices require longer preheating time).
-Select the corresponding detection mode (nucleic acid/protein).
2. Sample addition and testing
1. Clean the testing platform: Dip a small amount of ultrapure water or ethanol into dust-free paper and wipe the testing surface to avoid residual contamination.
2. Sample addition:
-NanoDrop instrument: Drop 1-2 µ L of sample onto the detection table and lower the detection arm.
-Colorimetric dish type instrument: Ensure that the colorimetric dish is clean and add an appropriate amount of sample (usually 50-200 µ L).
3. Reading: Click on "Measure" to record the data.
4. Cleaning: After the testing is completed, immediately clean the testing table with ultrapure water or ethanol to prevent sample crystallization from affecting subsequent testing.
3. Data recording and analysis
-Purity assessment of nucleic acid:
-DNA: A260/A280 ≈ 1.8 (pure DNA), A260/A230>2.0 (no pollutants).
-RNA: A260/A280 ≈ 2.0 (pure RNA).
-If the ratio is abnormal (such as A260/A280<1.6), it may indicate protein or phenolic contamination.
-Protein determination: When using Bradford or BCA methods, a standard curve needs to be prepared.
3、 Common problems and solutions
1. Abnormal test results (such as low A260/A280 ratio)
-Possible reasons: Protein or organic solvent contamination.
-Solution: Purify the sample again or use fluorescence quantification method (such as Qubit) instead.
2. Instrument error (such as "Sampletoo concentrated")
-Possible reason: The sample concentration exceeds the detection range.
-Solution: Dilute the sample and retest.
3. Baseline drift or instability
-Possible reasons: contamination of the testing platform or aging of the light source.
-Solution: Clean the testing table or contact an engineer to calibrate the light source.
4、 Practical skills
1. Avoid bubble interference: When adding samples, ensure that the droplets are intact and free of bubbles, otherwise it may cause fluctuations in absorbance.
2. Low concentration sample detection:
-Quantification using fluorescence method (such as Qubit) is more sensitive than UV method.
-If UV method must be used, the sample volume can be appropriately increased (e.g. 2 µ L → 3 µ L).
3. Long term storage recommendations:
-Turn on the device at least once a week to prevent the optical components from getting damp.
-Store in a dry and dust-free environment.