Relevance of purity ratios such as (A260/A230)
Purity ratios such as A260/A230 are often used as quick quality indicators for RNA and DNA preps. However, these values are not always predictive of downstream performance.
Low A260/A230 ratios typically indicate the presence of residual salts or buffer components (e.g., guanidine salts). While these can influence spectrophotometric readings, they do not necessarily inhibit downstream applications such as RT-PCR, qPCR or sequencing at the concentrations used after purification.
Purity ratios should be interpreted in context. If downstream applications perform well, suboptimal A260/A230 values do not necessarily mean that the protocol should be further modified.
In addition, spectrophotometric measurements (e.g., using low-volume spectrophotometers) are only reliable within a certain concentration range. For very low input samples or low nucleic acid concentrations, these measurements can be inaccurate and strongly influenced by background absorbance from buffers or residual contaminants. In such cases, fluorescence-based quantification methods are more suitable, and purity ratios derived from low-volume spectrophotometers should be interpreted with caution or may not be accurate.
If low purity ratios are observed together with poor downstream performance, additional wash steps or modified wash chemistry may help.
For more information, see our Application Note:
Effects of low A260/A230 ratios in RNA preparations on downstream applications.
Add additional wash steps or increase wash buffer volumes
One of the simplest ways to improve purity is to add an additional wash step or increase the wash buffer volume in existing steps. Additional or more extensive washing can help remove residual contaminants.
If there is bead clumping during washing, prolonged mixing can help resuspend the magnetic particles. You can alternate the mixing speed between a lower speed to cover the entire liquid volume and a higher speed at the well bottom to apply mechanical forces to the samples.
If wash buffers remain colored, an additional wash step using the same wash buffer may be sufficient to improve eluate quality. The parameters of the existing wash step can be used as a starting point.
If no visible abnormalities are observed in the lysate or wash buffers, the use of an alternative wash buffer – from a different Essential Kit – may improve eluate purity. Essential Kit C may be beneficial, as Buffer MW1 can help reduce inhibition in challenging sample materials.
Availability of stand-alone wash buffers
QIAsprint Connect workflows are modular, and most wash buffers are available as stand-alone components. You can check availability using the QIAsprint sets and consumables finder.
If additional wash steps do not have the desired effect, we recommend testing an alternative Essential Kit with different wash chemistry. While the lysis conditions defined by the PrepSet may be suitable, the wash chemistry of another Essential Kit may better address specific contaminants in the sample. See the Essential Kit overview.
Extend the air-dry time
Residual wash buffer left on the beads can negatively affect purity ratios and downstream performance. Many wash buffers contain ethanol, which is a well-known PCR inhibitor if carried over into the eluate. Extending the air-dry time after the final wash step allows improved evaporation of residual ethanol from the bead surface.
If there is significant ethanol carryover, there may be a detectable odor of ethanol in the eluate indicating insufficient drying.
At the same time, make sure not to over-dry the beads, as excessive drying can reduce elution efficiency and negatively impact nucleic acid recovery. Follow the guidance in Avoid overdrying the beads.
Consider the relationship between input amount and purity ratios
Very high input amounts can overload the binding capacity or wash efficiency, resulting in poorer purity values. Reducing the sample input amount often leads to significantly improved QC results. Follow the guidance in Consider input and sample limitations.
Inefficient sample disruption is another potential source of impurities in the eluate. Reducing the amount of input material or optimizing the sample disruption process can help. Extending incubation time for enzymatic digestion or, increasing the disruption time for applications that do not require high-molecular-weight DNA, can improve results.
Magnetic bead carryover
In general, residual magnetic beads in the elution plate do not negatively impact downstream applications. However, they may interfere with methods that rely on light scattering for sample analysis, such as measurements from low-volume spectrophotometers. In this case, make sure not to transfer the beads with the eluate into vessels used for downstream applications. To minimize bead carryover, reduce the input material. Follow the guidance in Consider input and sample limitations. System overloading can lead to incomplete removal of high amounts of proteins and other contaminants during the wash steps. Reducing the input amount can mitigate the bead carryover.