Troubleshooting Micro Volume Nucleic Acid Measurements on the Genova Plus/Nano
•A260 nm Absorbance maximum of nucleic acids
The optimal absorbance range for the accurate measurement of nucleic acids is between 0.050 and 1.000 Abs. When using a 1cm path length cuvette in the Genova Plus this absorbance range equates to concentrations of dsDNA of 2.5μg/ml to 50μg/ml. In the Genova Nano the equivalent dsDNA concentrations are 50μg/ml to 1000μg/ml @ 0.5mm path length and 125μg/ml to 2500μg/ml @ 0.2mm path length.
Q? Is the absorbance value between 0.050 and 1.000 @260nm?
NO – Dilute or concentrate sample.
YES – No corrections required.
When the measured absorbance values are low (<0.05) the contribution of instrument and sample preparation errors becomes too significant for accurate measurements to be made.
•A280 nm Absorbance maximum of proteins and phenol
280nm is the reference wavelength that is used when determining the purity of nucleic acid samples.
The ratio of the absorbance values at 260nm and 280nm for pure DNA is 1.8 and for pure RNA is 2.0. Lower values indicate that the sample is contaminated by proteins containing aromatic groups and/or phenol.
Q? Is the value approximately 1.8 for DNA and approximately 2.0 for RNA?
NO – Indicates that the sample may be contaminated by proteins or phenol. Purify the sample.
YES – Indicates that the sample is pure.
•A230 nm Absorbance maximum of carbohydrates
230nm is a second reference wavelength that is used when determining the purity of nucleic acid samples.
The ratio of the absorbance values at 260nm and 230nm for pure DNA and RNA should be greater than 2.0. Lower values indicate that the sample is contaminated by sugar, salts or organic solvents.
Q? Is the value greater than 2.0?
NO – Indicates that the sample may be contaminated by carbohydrates, salts or organic solvents. Purify the sample.
YES – Indicates that the sample is pure.
•A320 nm Measured to take account of turbidity in the sample solution
An absorbance measurement is taken at 320nm to correct for any turbidity in the solution when determining the concentration and purity of nucleic acid samples.
Is the value approximately 0.000?
NO – Indicates that particulate matter is present in the sample. Purify the sample or ensure that A320 correction is enabled.
YES – Indicates that the sample is free from particulate matter. A320 correction is not required.
•Frequent Sources of Errors
Dilution errors – Dilute the sample in the ratio 1:10 to 1:50 as greater ratios can lead to pipetting errors.
Pipettes – Check that the pipettes being used are clean, calibrated and functioning correctly.
Sample mixing – All nucleic acid samples should be vortexed prior to dilution and measurement to eliminate any concentration gradients in the sample.
Sample pH – The absorbance spectrum of nucleic acids is influenced by the pH value of the sample solvent, especially in solutions with low ionic strength. Use 10mM Tris-HCl pH 8.0 or TE pH 8.0 to buffer the sample.
Cuvette considerations – Quartz or special UV grade plastic cuvettes must be used for all nucleic acid sample measurements.
When using micro-volume plastic cuvettes, ensure that the instrument is fitted with the micro cuvette holder accessory, part code JY/637 071.
Plastic cuvettes should not be reused too often as UV light can degrade the plastic.
The recommend fill volume of the cuvette must be considered to eliminate any effect from the sample meniscus and stray light.
Air bubbles or other visible contaminations must be avoided.
Incorrect factor – Check that the correct multiplication factor/s have been entered into the instrument settings.
Calibration Filter/ Solution Check – Perform a measurement using a certified calibration solution. Re-calibrate the micro-volume accessory using the procedure described in the Genova Nano operating manual
An application note that contains the information detailed in this article, can be downloaded from the Jenway website by clicking on the following link:
The full range of Jenway products can be obtained from Camlab