Six kinds of common mistakes in the process of nucleic acid extraction by magnetic beads

Myth #1: The more magnetic beads are used, the better the extraction effect. Many teachers like to increase the amount of magnetic beads when the extraction is not effective. They think that adding magnetic beads can increase the number of nucleic acids. This idea is not desirable. The main characteristic of magnetic beads is that they can be dispersed in liquids or separated by solid state and liquid phase under the action of an external magnetic field. The ratio of any reagent system, magnetic beads, and liquids should have a certain threshold, exceeding a certain proportion. Too many magnetic beads will lose their dispersion characteristics because they cannot be uniformly dispersed in the liquid, and the efficiency of contact between the nucleic acid beads and the liquid cannot be sufficiently increased during the washing process. Excessive magnetic beads also absorb more impurities and have a great influence on the effect of impurity removal. Even sometimes, too many magnetic beads adsorb proteases, lysozyme, etc., which play a major role in the liquid system, resulting in inefficiency of the entire kit. There are many times when the extraction effect is not good, reducing the use of magnetic beads, but on the contrary is the best way to enhance the extraction effect. Generally, the amount of reference magnetic beads given by the magnetic bead kit is a slight excess. Therefore, it is not necessary to increase the amount of magnetic beads to improve the adsorption efficiency. However, if it is determined that the amount of magnetic beads is insufficient, the extraction effect is not sufficient. Well, it is possible to improve the extraction effect by increasing the amount of magnetic beads within a certain range. Take GNT-02 series magnetic beads as an example, when taking constant samples (plant tissue, whole blood, etc.), the dosage is usually 10ul/time; when extracting trace samples (such as serum free DNA, buccal swab, etc.), magnetic beads The dosage is 15~20ul/time. If you need to exceed this amount, you need to communicate with the technical engineer.

Misunderstanding 2: The more reagents are used, the better the extraction effect

Cracking effect is not good? Add more lysis solution. Bad washing effect? Add more wash solution. This is inertial thinking that many customers use in kits. However, for the magnetic beads method, each additional part of the liquid volume reduces the probability of more magnetic beads colliding, and decreasing the magnetic beads collision probability will result in a significant decrease in the adsorption rate. So many times, although the increase of lysing and washing liquids can indeed play a role in enhancing the cracking and enhancing the washing, the core of the magnetic beads extraction method is the efficiency of magnetic beads adsorption of nucleic acid, and cannot ensure that the magnetic beads collision efficiency cannot guarantee the efficiency of nucleic acid extraction. Therefore, simply increasing the amount of reagent used to improve the extraction effect may not be completely effective. For the GNT-B02 whole blood genomic DNA kit, the amount of lysate used should not exceed 400 ul/time, and the amount of wash solution should not exceed 500 ul/time. If you really need to scale up the system, the amount of magnetic beads and sample should also increase accordingly. This amplification is not necessarily proportional.

Misunderstanding 3: The more washing times, the better the extraction effect

When the extracted nucleic acid impurities are too much, the user may consider washing it several times to obtain more pure nucleic acid. Increasing the number of washings is indeed beneficial for purifying nucleic acids, but considering that each wash will lose a certain amount of nucleic acid and increase the possibility of nucleic acid cleavage hydrolysis, it is generally desirable to control the number of washings in 2 to 4 times. The GNT series of kits, single purification kits were washed twice, plant and animal kits were washed three times, and blood kits were washed three or four times.

Misunderstanding 4: The more samples are taken, the better the effect of extraction

In cases where the sample is not fresh enough or the nucleic acid content itself is very small, the nucleic acid extraction effect is not good, and many teachers will use multiple sampling methods to increase the amount of nucleic acid extraction. However, simply increasing the sample size will sometimes introduce too much impurities, which will exceed the cracking capacity of the lysate and will also reduce the extraction efficiency. Therefore, it is not recommended to simply increase the sample size to increase the extraction volume. If it is indeed due to a lack of sample size and the extraction volume is too low, it is recommended that the pretreatment be enriched or concentrated prior to the extraction. Or increase the completeness of the lysis so that more nucleic acids are exposed is also a solution.

Myth 5: Some kind of magnetic beads is good, it should be effective in all tests.

The types of magnetic beads are various, particle size is different, dispersion is different, magnetic response time is different, different substrates are coated, the outer layer is modified with different functional groups, the coating density is different, and the functional group arm length is different, which will result in magnetic beads. Characteristics vary widely. Therefore, different magnetic beads are different in their experiments and systems. Just like reagents for nucleic acid extraction, the formulation is not exactly the same. The same applies to magnetic beads for nucleic acid extraction. The properties are not exactly the same. Some magnetic beads exhibit higher adsorption efficiency in the extraction of macronuclides, and some are more suitable for micronucleic acid extraction. Some magnetic beads are suitable for some acidic series reagent systems, and some magnetic beads are suitable for alkaline series reagent systems. Some magnetic beads have good magnetic response but fast settling speed and are more suitable for magnetic rod type automatic extractor. Some magnetic beads have slow sedimentation speed but long magnetic response time, and are more suitable for pipetting type automatic extractor. Few magnetic beads can be applied to all experiments. In addition to fixed kits, in most cases, magnetic beads and reagent systems need to be adjusted for a certain period of time.

Misunderstanding 6: Contrasting with a kit is not good, it is bad magnetic beads

Many customers in the process of screening magnetic beads are already in the mature reagent system and simply replace magnetic beads in equal amounts to compare magnetic bead effects. This will easily lead to the conclusion that some kind of magnetic beads are not effective, but in fact, because the systems and dosages of different magnetic beads are different, it is often necessary to adjust them to obtain better extraction results. In addition to producing magnetic beads and kits, Keygen Biotech also provides magnetic bead extraction technology services, including helping customers adjust the amount of magnetic beads and the details of the reagents, allowing customers to find more suitable systems to obtain the best experimental results.