In the past two years, as pathogen detection has become more popular, pathogen nucleic acid detection has gradually entered people’s field of vision and has become the gold standard for pathogen detection. Faced with large quantities of samples, testing results need to be issued in a short period of time, and people’s needs for fast and efficient testing methods are becoming more and more urgent. As an important step of nucleic acid detection, nucleic acid extraction also faces the requirements of throughput and high efficiency. Automatic nucleic acid extraction (magnetic bead method nucleic acid extraction) has been quickly promoted and applied because of its fast, efficient, easy-to-use, safe and non-toxic characteristics, and has become a standard in laboratories. However, during the extraction process, certain factors will affect the extraction results and require the operator’s attention. The editor of Aisen has compiled the relevant knowledge of the factors affecting nucleic acid extraction by magnetic bead method, and let’s share it with you.
Magnetic bead method nucleic acid extraction consists of lysis, washing and elution steps. The factors that affect these steps will also affect the extraction efficiency of magnetic beads. Below we explain each step one by one.
Factors affecting the lysis step
1.Lysis solution composition
For some samples that are difficult to lyse, the lysis ability of the lysate can be enhanced by increasing the concentration of guanidine salt and surfactant. Guanidine salts and surfactants can promote protein denaturation, destroy cell membranes, dissociate nucleic acids and proteins, and release more nucleic acids.
2.pyrolysis temperature and time
Increasing the temperature and increasing the lysis time can make the cell lysis more thoroughly and get more nucleic acids. You need to explore the appropriate temperature and time; too high temperature or too long time may also lead to nucleic acid fragmentation or reduced extraction efficiency caused by alcohol volatilization.
3.magnetic beads adsorption capacity
magnetic bead extraction series magnetic beads are generally silanol magnetic beads, and the adsorption capacity of the magnetic beads is affected by the specific surface area of the magnetic beads and the density of the modified group. Generally, magnetic beads with more modified groups and larger specific surface area are selected. Generally, the smaller the particle diameter of the magnetic beads, the larger the specific surface area. But it is not that the smaller the particle size of the magnetic beads, the better, the smaller the particle size of the magnetic beads, the adsorption speed will also slow down, you can choose according to the actual sample situation. The binding buffer is generally combined with isopropanol or ethanol. Generally, the binding capacity can be enhanced by increasing the ratio of alcohol, and the concentration of chaotropic salt should be increased to promote the binding of nucleic acid and magnetic beads.
Factors affecting washing steps
Washing is very important for the entire extraction step. It is related to the purity of nucleic acid, which directly affects downstream PCR applications. The oscillation amplitude and mixing time of the magnetic bar will affect the washing efficiency.
1.Magnetic rod oscillation amplitude
If the oscillation amplitude of the magnetic rod is too small, there will be impurities remaining on the magnetic beads, which will affect the ability of the magnetic beads to adsorb nucleic acids; if the oscillation is too violent, part of the nucleic acid will be lost or the nucleic acid will be broken or degraded, and the yield will decrease.
If the mixing time is too short, there will be impurities remaining on the magnetic beads, which will affect the ability of the magnetic beads to adsorb nucleic acids; if the mixing time is too long, part of the nucleic acid will be lost or the nucleic acid will be broken or degraded, and the yield will decrease.
The influence of elution step factors
The elution step directly affects the yield of nucleic acid. The factors affecting the elution efficiency are the drying degree of the magnetic beads and the elution temperature and time.
1.Dryness degree of magnetic beads
If the magnetic beads are not fully dried, there may be impurities such as alcohol, which will affect the subsequent PCR reaction; if the magnetic beads are too dry, the nucleic acid may dry up on the magnetic beads, resulting in a decrease in elution efficiency.
2.elution temperature and time
Appropriately increasing the elution temperature and time will help the nucleic acid to be released from the magnetic beads. If the time is too long or the temperature is too high, the nucleic acid may be degraded, and the balance needs to be grasped.
The relevant content about the factors that affect the magnetic bead method of nucleic acid extraction is the above. When performing the magnetic bead method of nucleic acid extraction, ensure that the above factors are reasonable, so that the extraction quality can be better. Aisen Biotechnology has extensive experience in the production of nucleic acid extraction systems and nucleic acid extraction kits. It uses high-quality technology and reliable quality. Customers in need are welcome to call for consultation.