Magnetic bead method of nucleic acid extraction steps and principles
With the continuous development of biomedicine, nucleic acid extraction technology is also being updated. Magnetic bead nucleic acid extraction, as a new type of nucleic acid extraction technology, can extract nucleic acids with high throughput and automation, meeting the needs of today’s biotechnology. In addition, the steps of magnetic bead method for nucleic acid extraction are relatively simple, easy to operate, and are well received. Next, I will share with you the steps and principles of magnetic bead method for nucleic acid extraction.
Magnetic bead method for nucleic acid extraction steps
1. Add 1 mL of deionized water to each bottle of 40mg proteinase K dry powder to a final concentration of 40mg/mL, and add 500 μL of deionized water to each bottle of 20mg proteinase K dry powder to a final concentration of 40mg/mL, and mix it upside down. It can be completely dissolved once, and it can be stored at 4℃ for short-term use. Please keep it at -20℃ for long-term storage. Repeated freezing and thawing should not exceed five times.
2. The tissue sample is ground into fine powder with 10-30 mg of liquid nitrogen, transferred to a 1.5 mL centrifuge tube, resuspended in 200 μL of 1 X PBS buffer, and then proceeded to the next step. The liquid sample goes directly to the next step.
3. Take 500μL of virus lysate VL and add it to a 1.5mL enzyme-free centrifuge tube, then add 25μL of proteinase K and 25μL of viral magnetic beads (shaken well before adding), and then add 200μL of serum/plasma/tissue homogenate sample , Vortex and mix well, 55% C water bath for 20 minutes, invert and mix for 10 seconds every 5 minutes.
4. Take out the enzyme-free centrifuge tube in the water bath, place it on the magnetic stand, and magnetize for 90 seconds to completely adsorb the magnetic beads. Use a pipette to carefully aspirate and discard the supernatant (be careful not to attract the magnetic beads at the bottom of the tube).
5. Remove the centrifuge tube from the magnetic stand, add 700μL of rinsing solution BufferA, mix by pipetting or vortex for 30 seconds to resuspend the magnetic beads, then place the centrifuge tube on the magnetic stand, and magnetize for 90S to make the beads Adsorbed completely, carefully aspirate and discard the supernatant with a pipette.
6. Take 700μL of rinsing solution BufferB, add it to the centrifuge tube, try not to blow off the magnetic beads, and then directly aspirate and discard the supernatant.
7. Add 80μL of elution buffer, remove the centrifuge tube, and bathe in a water bath at 56°C for 5 min. During this period, vortex 3-4 times to elute the nucleic acid from the magnetic beads.
8. Place the centrifuge tube on the magnetic stand and magnetically attract for 60 seconds, transfer the liquid to a new 1.5mL enzyme-free centrifuge tube, and proceed directly to the downstream experiment or store at -20°C.
The principle of magnetic bead method for nucleic acid extraction
Its principle mainly relies on electrostatic interaction, hydrophobic interaction and hydrogen bonding interaction. The DNA/RNA in the cell or tissue is released under the action of the lysate. At this time, the surface-modified superparamagnetic silica nanomagnetic beads “specifically bind” with nucleic acid to form a “nucleic acid-magnetic bead complex”. Then under the action of an external magnetic field, the complex is separated. Then the eluate is used to wash away non-specifically adsorbed impurities, desalt and purify to obtain the nucleic acid substance to be extracted.