Common misconceptions during nucleic acid extraction by magnetic bead method

The extraction of nucleic acid using magnetic beads is still a relatively new nucleic acid extraction method, compared with the traditional chloroform isoamyl alcohol extraction method and spin column kit method. This method is still not understood well by many people, and there are some misunderstandings in the process of using magnetic bead method to purify nucleic acids.

Misconceptions 1

The more magnetic beads, the better the extraction effect

Many researchers like to increase the amount of magnetic beads when the extraction effect is not good, thinking that more magnetic beads can extract more nucleic acids, and we have to say that this idea is not desirable. The main characteristic of magnetic beads is that they can be dispersed in the liquid, and also separated from the liquid phase in a solid state under the action of the applied magnetic field. In every reagent system, the ratio of magnetic beads to liquid has a certain threshold, beyond a certain ratio, too many magnetic beads will lose their dispersion characteristics because they cannot be evenly dispersed in the liquid, which also makes the washing process unable to fully increase the efficiency of the contact between magnetic beads and liquid of nucleic acid. Excessive magnetic beads will also adsorb more impurities, which will have a bad impact on the effect of purification. Sometimes when too many magnetic beads adsorb functional components that play a major role in the liquid system such as protease and lysozyme, resulting in inefficiency of the whole kit. When the extraction effect is poor, reducing the amount of magnetic beads used is instead the optimal way to improve the extraction effect. Usually, the reference bead dosage given by the magnetic bead method kit is slightly excessive, so there are not many cases when it is necessary to increase the bead dosage to improve the adsorption efficiency, but if it is determined that the extraction effect is poor due to insufficient bead dosage, it is possible to improve the extraction effect by increasing the bead dosage within a certain range.

Misconceptions 2

The more reagents you use, the better the extraction effect

Bad lysis? Add more lysis solution. Bad washing? Add more washing solution. This is the usual thinking of many customers in using kits. But for the magnetic bead method, every increase in liquid volume reduces the chance of more bead collisions, and reducing the chance of bead collisions will lead to a significant decrease in adsorption rate. Therefore, in many cases, although increasing the lysis solution and washing solution can indeed play the role of enhancing lysis and enhancing washing, the core of magnetic bead method extraction is the efficiency of magnetic bead adsorption of nucleic acids, and the inability to guarantee the efficiency of magnetic bead collision cannot guarantee the efficiency of nucleic acid extraction, so simply increasing the amount of reagents used to improve the extraction effect is not necessarily fully effective. If you really need to amplify the system, the amount of magnetic beads and samples should be increased accordingly, and this amplification is not necessarily in equal proportion.

Misconceptions 3

The more washing, the better extraction yield.

When the extracted nucleic acid has too many impurities, users may consider to perform more washes to obtain a purer nucleic acid. It is true that increasing the number of washes is good for purifying nucleic acids, but considering that each wash loses a certain amount of nucleic acids and increases the possibility of nucleic acid breakage and hydrolysis, it is generally appropriate to limit the number of washes to 2-4 times.

Misconceptions 4

The more samples you extract, the better the extraction yield

In cases where the sample is not fresh enough or the nucleic acid content is very small, the nucleic acid extraction is often not effective, and many researchers use more samples to increase the amount of nucleic acid extracted. However, simply increasing the sample size can sometimes introduce too many impurities that exceed the lysis capacity of the lysate and also reduce the extraction efficiency, so it is not recommended to increase 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 go through an enrichment or concentration step before starting the extraction in the pretreatment. Alternatively, increasing the completeness of the lysis so that more nucleic acids are exposed is also a solution.

Misconceptions 5

If one kind of magnetic beads is good, it should work well in all tests.

There are various types of magnetic beads, with different particle size, different dispersion, different magnetic response time, different encapsulated base matrix, different outer modified functional groups, different encapsulation density, and different functional group arm length, which will lead to a wide variety of bead properties. Therefore, the experiments and systems to which different magnetic beads are adapted are also different. Just like the same reagents for nucleic acid extraction, the formulations are not exactly the same, the same magnetic beads applied to nucleic acid extraction, the properties are not exactly the same. Some magnetic beads show higher adsorption efficiency in macro-nucleic acid extraction, while some are more suitable for micro nucleic acid extraction. Some beads are suitable for acidic series reagent systems, while others are suitable for basic series reagent systems. Some magnetic beads have good magnetic response but fast settling speed, which is more suitable for magnetic rod type automatic extractor; some magnetic beads have slow settled speed but long magnetic response time, which is more suitable for pipetting type automatic extractor. There is seldom one kind of magnetic beads can be suitable for all experimental situations, except for the fixed reagent kit match, in most cases, the magnetic beads and reagent system have to do some time to adjust the match.

Misconceptions 6

Poor comparison with a certain kit means that the beads are not good

Many laboratories in the process of screening magnetic beads simply replace the beads in equal amounts under the mature reagent system and use it to compare the effect of magnetic beads.

In this way, it is easy to conclude that a certain magnetic bead is not effective. But in fact, because different magnetic beads are suitable for different systems and dosage is different, the beads often need to be adjusted to get better extraction effect.