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Compared with the traditional chloroform isoamyl alcohol extraction method and spin column kit method, the use of biological magnetic beads for nucleic acid extraction is a novel method for nucleic acid extraction. However, there are still many people who don’t fully understand this method. There are also some misunderstandings in the process of purifying nucleic acids by magnetic beads.
Myth 1: The more magnetic beads are used, the better the extraction effect
Many people like to increase the number of magnetic beads when the extraction results are not good. They think that adding more magnetic beads can adsorb more nucleic acids. But this is not always the case.
The main feature of magnetic beads is that they can be dispersed in a liquid, and can be separated from the liquid phase in a solid-state under the action of an external magnetic field. In any reagent system, the ratio of magnetic beads to liquid should have a certain threshold. Beyond a certain ratio, too many magnetic beads will lose their dispersion characteristics because they cannot be uniformly dispersed in the liquid, which also makes it impossible to fully increase the efficiency of nucleic acid, magnetic beads, and liquid contact during the washing process. Excessive magnetic beads will also adsorb more impurities, making it more difficult to remove impurities. Moreover, too many magnetic beads will adsorb protease, lysozyme and other functional components that play a major role in the liquid system, resulting in low extraction efficiency of the kit. Many times, when the extraction effect is not good, reducing the number of magnetic beads used is the best way to improve the extraction effect.
Under normal circumstances, the amount of reference magnetic beads given by the kit is slightly excessive. Therefore, it is not necessary to increase the amount of magnetic beads to improve the adsorption efficiency. However, if it is determined that the extraction effect is not good due to the insufficient amount of magnetic beads. Then, it is possible to improve the extraction effect by increasing the number of magnetic beads within a certain range.
Myth 2: The more reagents are used, the better the extraction effect
Poor lysing effect? Add more lysate. Poor washing effect? Add more wash solution. This is the inertia of many customers in using kits.
However, for the magnetic bead method, each increase in the volume of liquid reduces the probability of collision of magnetic beads, and reducing the probability of collision of magnetic beads will cause a large decrease in the adsorption efficiency. So in many cases, although adding lysis buffer and washing buffer can indeed enhance the lysis and enhance the washing effect, the core of magnetic bead extraction is the adsorption efficiency of the magnetic bead for nucleic acid. A lower collision between magnetic beads results in the lower extraction effect. Therefore, simply increasing the amount of reagent used to improve the extraction effect may not be completely effective.
Myth 3: The more washing times, the better the extraction effect
When there are too many impurities in the extracted nucleic acid, the user will consider performing more washing to obtain more pure nucleic acid. Increasing the number of washings is indeed beneficial for the purification of nucleic acids, but it is considered that a certain amount of nucleic acids is lost with each washing, and the possibility of nucleic acid fragmentation and hydrolysis is increased. Therefore, the number of washing times should be controlled at 2 to 4 times.
Myth 4: The more samples you add, the better the extraction effect
When the sample is not fresh enough or the nucleic acid content itself is very small, the nucleic acid extraction is often not good. At this time, many people will increase the amount of nucleic acid extraction by adding samples.
But simply increasing the sample size sometimes introduces excessive impurities. In addition, exceeding the lysing capacity of the lysis buffer will also reduce the extraction efficiency, so it is not recommended to increase the amount of extraction by simply increasing the sample size.
If the extraction volume is indeed too low due to insufficient sample volume, it is recommended to go through the enrichment or concentration step before starting the extraction during pretreatment. Or increasing the intensity of lysis to expose more nucleic acids is also an alternative.
Myth 5: If a certain kind of magnetic bead is good, it should work well in all tests
There are various types of magnetic beads, with different particle sizes, different dispersions, different magnetic response times, different coating base matrices, different modified functional groups on the surface, different coating densities, and different functional arm lengths, all of which will lead to different characteristics of magnetic beads. Therefore, different magnetic beads adapt to different experiments and systems.
Some magnetic beads show higher adsorption efficiency in constant nucleic acid extraction, and some magnetic beads are more suitable for trace nucleic acid extraction. Some magnetic beads are suitable for acidic series reagent system, some magnetic beads are suitable for alkaline series reagent system. Some magnetic beads have a good magnetic response but fast sedimentation speed, which is more suitable for magnetic rod type automatic extraction instrument; some magnetic beads have slow sedimentation speed but long magnetic response time, and are more suitable for pipette type automatic extraction instrument.
Few magnetic beads are suitable for all experimental situations. Except for fixed reagent kits, in most cases, a series of adjustments must be made to the magnetic beads and reagent system. In addition to magnetic beads and kits, CD Bioparticles also provides beads based kits development services, including helping clients adjust the amount of magnetic beads and the details of reagents, so that clients can find a more suitable system to obtain the best experimental results.