Fine-tune your nucleic acid yield and purity

Quantification of nucleic acids is an important step during your isolation of DNA or RNA. Below we describe step by step where to fine-tune your purification and where you need to put your attention to.

Factor influencing your sample purity and yield

• Sample quality and preparation
• Sample amount and storage conditions
• Carry over of humic substances from the sample disruption
• Carry over of ethanol, salts or humic substances from the wash buffer

Sample preparation

• Low sample input may lead to a decrease in the A₂₆₀/A₂₈₀ ratios
• Overestimation of DNA yield may be caused by RNA contaminations presence in the sample

• Use a specialized kit such as a XS version that will concentrate your nucleic acid
• If RNA is still present you may want to check your sample quality and storage conditions

Sample disruption

• Proteins, phenol or humic substances from the disruption of your samples may lead to a decrease of A₆₀/A₂₃₀ and A₂₆₀/A₂₈₀
• Excess amounts of RNA during DNA isolation may lead to an increase of the A₂₆₀/A₂₈₀ ratio

• Modify your sample disruption e.g. increase or decrease time, change beads
• Change the incubation time with Proteinase K
• Check for soil samples if there are variations of the lysis buffer available
• Check your sample storage conditions and quality

Sample washing

• Ethanol or salts like thiocyanate, sodium or potassium that are present in the wash buffer if carried over to the sample may lead to a drecreased A₂₆₀/A₂₃₀
• Presence of humic substances from soil samples or secondary plant metabolites that are not removed during the washing steps may lead to an increased A₂₆₀/A₂₃₀ ratio

• Ensure your silica membrane is dry after each wash and spin step
• Try to avoid to overload your column, change the collection tube after each spin
• Check the colour of your sample, maybe introduce another wash step

Sample detection

• Using the wrong blank for your sample will lead to an increase of your A₂₆₀/A₂₃₀ ratio, changing the blank will bring the ratios back to a normal level
• If your blank is contaminated with e.g. traces of chaotropics salts or similar this will lead to an increase in your A₂₆₀/A₂₃₀ ratio

• Use blank solution with same pH and composition as your samples
• Change the pipet tip after each step

Summary

In this overview we summerize the points mentioned in the above section. If you have any further questions please contact bio-tech@mn-net.com or use our
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