Six Common Misunderstandings in the Process of nucleic acid extraction by magnetic bead Method

Article source: Super Lab, IVD Sharing Library

Many teachers like to increase the amount of magnetic beads when the extraction effect is not good, believing that adding more magnetic beads can absorb more nucleic acids. It must be said that this idea is not advisable. The main feature of magnetic beads is that they can not only be dispersed in liquids but also be separated from the liquid phase in a solid state under the action of an external magnetic field. For any reagent system, the ratio of magnetic beads to liquid should have a certain threshold. If the ratio exceeds a certain limit, too many magnetic beads will lose their dispersion characteristics due to the inability to be uniformly dispersed in the liquid. This also makes it impossible to fully increase the efficiency of contact between the nucleic acid magnetic beads and the liquid during the washing process.

Excessive magnetic beads will also adsorb more impurities, which has a significant impact on the effect of impurity removal. Sometimes, an excessive number of magnetic beads can adsorb functional components such as protease and lysozyme that play a major role in the liquid system, leading to low efficiency of the entire reagent kit. There are many times when the extraction effect is not good, reducing the usage of magnetic beads is actually the best way to improve the extraction effect.

Under normal circumstances, the reference amount of magnetic beads provided by the magnetic bead method kit is usually slightly excessive. Therefore, it is not common to increase the amount of magnetic beads to improve the adsorption efficiency. However, if it is determined that the poor extraction effect is caused by insufficient amount of magnetic beads, the extraction effect can be improved within a certain range by increasing the amount of magnetic beads.

Poor cracking effect? Add more lysis buffer. The washing effect is not good? Add more detergent. This is the habitual thinking of many customers when using the reagent kits.

However, for the magnetic bead method, every increase in the volume of the liquid reduces the probability of magnetic bead collisions to a greater extent, and a decrease in the probability of magnetic bead collisions will lead to a significant drop in the adsorption rate. Therefore, in many cases, although increasing the lysis buffer and washing buffer can indeed enhance the lysis and washing effects, the core of magnetic bead extraction lies in the efficiency of magnetic bead adsorption of nucleic acids. If the collision efficiency of magnetic beads cannot be guaranteed, the efficiency of nucleic acid extraction cannot be guaranteed. Thus, simply increasing the amount of reagent used to improve the extraction effect is not necessarily completely effective.

When the extracted nucleic acid contains too many impurities, users will consider washing it several times to obtain a purer nucleic acid. Increasing the number of washes is indeed beneficial for purifying nucleic acids. However, considering that each wash incurs a certain amount of nucleic acid loss and increases the possibility of nucleic acid cleavage and hydrolysis, it is generally advisable to control the number of washes at 2 to 4 times.

When the samples are not fresh enough or the nucleic acid content is already very low, the nucleic acid extraction effect is often not good. Many teachers will adopt the method of taking multiple samples to increase the amount of nucleic acid extracted. However, simply increasing the sample size sometimes introduces excessive impurities, exceeding the pyrolysis capacity of the lysis buffer, and also reduces the extraction efficiency. Therefore, it is not recommended to achieve the purpose of increasing the extraction volume by simply increasing the sample size.

If the extraction volume is indeed too low due to insufficient sample size, it is recommended to first carry out enrichment or concentration steps in the pretreatment process before starting the extraction. Or increasing the completeness of lysis to expose more nucleic acids is also a solution.

The types of magnetic beads are diverse. Different particle sizes, dispersibility, magnetic response times, coating base matrices, outer modification functional groups, coating densities, and functional group arm lengths can lead to a wide range of magnetic bead properties. Therefore, different magnetic beads are suitable for different experiments and systems. Just like the reagents used for nucleic acid extraction, their formulas are not exactly the same, and the magnetic beads applied in nucleic acid extraction also have different properties.

Some magnetic beads exhibit higher adsorption efficiency in the extraction of macro nucleic acids, while others are more suitable for the extraction of trace nucleic acids. Some magnetic beads are suitable for slightly acidic reagent systems, while others are suitable for slightly alkaline reagent systems. Some magnetic beads have good magnetic response but a fast sedimentation rate, making them more suitable for magnetic rod-type automatic extractors. Some magnetic beads have a slow sedimentation rate but a long magnetic response time, making them more suitable for pipetting automatic extraction instruments. Few magnetic beads can be used in all experimental situations. Apart from the fixed reagent kits, in most cases, the magnetic beads and the reagent system need to be adjusted for a certain period of time.

Many customers, during the process of screening magnetic beads, simply replace the magnetic beads in equal amounts under a mature reagent system to compare the effects of the magnetic beads.

This would easily lead to the conclusion that a certain type of magnetic bead is not effective. However, in reality, since different magnetic beads are suitable for different systems and dosages, adjustments are often needed to achieve better extraction results.