磁珠法植物基因组 DNA 提取试剂盒为植物样品的 DNA 快速制备提供了一个简单快速的解决方案。试剂盒基于超顺磁性的磁性粒子纯化技术，提取过程中无需使用有毒的酚氯仿抽提，也无需进行耗时的醇类沉淀，整个提取过程只需 40 分钟左右。该试剂盒可整合磁棒法或移液法自动核酸提取仪进行高通量提取实验，也可使用磁力分离架进行手工操作。
Magnetic 纯化系统以超顺磁性的纳米磁性粒子为基质，这种磁性粒子在高浓度离液剂的 条件下可通过氢键和静电特异的吸附核酸，而蛋白质或其它非特异吸附的少量杂质经洗涤被去除，最后用低盐缓冲液或 RNase Free ddH₂O 洗脱核酸。纯化的核酸可适用于各种常规操作，包括 PCR、荧光定量 PCR 和病毒检测等各种下游实验。
产品组分表：
| D1372310 | Component | 50 T | Storage | | D1372310A | Buffer MLP1 | 30 ml | RT | | D1372310B | Buffer MPN | 30 ml | RT | | D1372310C | Buffer RW1P | 35 ml | RT | | D1372310D | Buffer MWP | 65 ml | RT | | D1372310E | Buffer EB | 15 ml | RT | | D1372310F | 磁珠XT | 2.2 ml | RT | | D1372310G | RNase A (10mg/ml) | 300 µl | RT |
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储存条件
试剂盒中所有组分可在室温干燥保存15个月。Buffer MLP1在低温下可能会有沉淀析出，可在37℃水浴中重新溶解，摇匀后使用。
提取得率
植物材料 | 提取量 | 平均 DNA 产量 | OD260/OD280 | 小麦 | 100 mg | 18-25 µg | 1.7-1.9 | 松树 | 100 mg | 25-30 µg | 1.7-1.9 | 马铃薯 | 100 mg | 4-6 µg | 1.7-1.9 | 番茄 | 100 mg | 10-15 µg | 1.7-1.9 | 烟草 | 100 mg | 10-15 µg | 1.7-1.9 | 水稻 | 100 mg | 10-25 µg | 1.7-1.9 | 大豆 | 100 mg | 10-25 µg | 1.7-1.9 | 玉米 | 100 mg | 20-30 µg | 1.7-1.9 | 棉花 | 100 mg | 10-25 µg | 1.7-1.9 |
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操作步骤
1. 植物材料破碎
1) 前处理方案1：向 2 mL 离心管中加入 1 颗钢珠，50–100 mg新鲜植物材料或 20 mg干燥植物材料，液氮预冷后 40 Hz，30 sec充分研磨成粉末，加入 400 µL Buffer MLP1 和 5 µL RNase A (10 mg/mL)，迅速颠倒混匀后，瞬时离心，70 °C金属或水浴 10 min，间隔 5 min取出手动混匀一次。
2) 前处理方案2：称取经液氮充分研磨好的新鲜植物材料粉末约 50–100 mg或干燥植物材料粉末约 15–20 mg，迅速转移到预先装有 400 µL Buffer MLP1 和 5 µL RNase A (10 mg/mL) 的 2 mL离心管中，迅速颠倒混匀后，瞬时离心，70 °C金属或水浴 10 min，间隔 5 min取出手动混匀一次。
2. 14,000 rpm离心 4 min，转移 300 µL上清至新的 1.5 mL离心管中。
3. 加入 550 µL Buffer MPN 和 40 µL磁珠 XT，涡旋振荡混匀 5 sec后将离心管放于 25 °C、1500 rpm的恒温混匀仪上振荡混匀 5 min或将离心管连续颠倒混匀 5 min。
4. 将离心管放置于磁力架上静置 1 min，待磁珠完全吸附后，彻底弃去溶液（保持离心管固定于磁力架上）。
5. 将离心管从磁力架上取下，加入 600 µL Buffer RW1P后涡旋点震 1 min或涡旋振荡 5 sec后放入 25 °C、1500 rpm的恒温混匀仪上振荡混匀 2 min（振荡过程中确保磁珠处于混匀状态）。之后将离心管放于磁力架上静置 1 min，待磁珠完全吸附于离心管侧壁后轻轻颠倒磁力架将离心管盖上的杂质洗落后彻底弃去溶液（保持离心管固定于磁力架上）。
6. 将离心管从磁力架上取下，加入 600 µL Buffer MWP后涡旋点震 1 min或涡旋振荡 5 sec后放入 25 °C、1500 rpm的恒温混匀仪上振荡混匀 2 min（振荡过程中确保磁珠处于混匀状态）。之后将离心管放于磁力架上静置 1 min，待磁珠完全吸附于离心管侧壁后轻轻颠倒磁力架将离心管盖上的杂质洗落后彻底弃去溶液（保持离心管固定于磁力架上）。
7. 重复步骤 6。
8. 保持离心管固定于磁力架上，用移液器进一步去除离心管管底和管盖上的溶液，之后室温放置 3–5 min，使乙醇挥发干净。
9. 将离心管从磁力架上取下，加入 50–100 µL Buffer EB。涡旋振荡使磁珠完全悬浮于洗脱液中后将其放于 65 °C、1500 rpm的恒温混匀仪上振荡洗脱 5 min。
10. 将离心管转移至磁力架上吸附 2 min，待磁珠完全吸附后，小心将核酸溶液转移至一个新离心管（自备）中，并于适当条件保存。
The Magnetic Plant DNA Kit provides a simple and rapid solution for DNA preparation from plant samples. The kit is based on superparamagnetic particle purification technology and eliminates the need for toxic phenol-chloroform extraction or time-consuming alcohol precipitation. The entire extraction process takes approximately 40 minutes. The kit can be integrated with automated nucleic acid extraction instruments using magnetic rod-based or pipetting-based methods for high-throughput extraction, or used manually with a magnetic separation rack.
The magnetic purification system uses superparamagnetic nanoparticles as the purification matrix. Under high concentrations of chaotropic salts, these magnetic particles specifically adsorb nucleic acids through hydrogen bonding and electrostatic interactions. Proteins and other minor nonspecifically adsorbed impurities are removed through washing. Finally, the purified nucleic acids are eluted using a low-salt buffer or RNase-Free ddH₂O. The purified nucleic acids are suitable for various routine applications, including PCR, quantitative real-time PCR (qPCR), virus detection, and other downstream experiments.
Product Components
D1372310 | Component | 50 T | Storage |
|---|
D1372310A | Buffer MLP1 | 30 mL | RT |
D1372310B | Buffer MPN | 30 mL | RT |
D1372310C | Buffer RW1P | 35 mL | RT |
D1372310D | Buffer MWP | 65 mL | RT |
D1372310E | Buffer EB | 15 mL | RT |
D1372310F | Magnetic Beads XT | 2.2 mL | RT |
D1372310G | RNase A (10 mg/mL) | 300 µL | RT |
Storage Conditions
All kit components can be stored at room temperature in a dry environment for 15 months. Buffer MLP1 may precipitate at low temperatures. If precipitation occurs, warm the buffer in a 37 °C water bath until the precipitate has completely dissolved. Mix thoroughly before use.
DNA Yield
Plant Material | Sample Amount | Average DNA Yield | OD260/OD280 |
|---|
Wheat | 100 mg | 18–25 µg | 1.7–1.9 |
Pine | 100 mg | 25–30 µg | 1.7–1.9 |
Potato | 100 mg | 4–6 µg | 1.7–1.9 |
Tomato | 100 mg | 10–15 µg | 1.7–1.9 |
Tobacco | 100 mg | 10–15 µg | 1.7–1.9 |
Rice | 100 mg | 10–25 µg | 1.7–1.9 |
Soybean | 100 mg | 10–25 µg | 1.7–1.9 |
Corn | 100 mg | 20–30 µg | 1.7–1.9 |
Cotton | 100 mg | 10–25 µg | 1.7–1.9 |
Protocol
1. Plant Material Disruption
1) Pretreatment Method 1: Add one steel bead and 50–100 mg of fresh plant material or 20 mg of dried plant material to a 2 mL centrifuge tube. Pre-cool the tube with liquid nitrogen, then grind the sample thoroughly into a fine powder at 40 Hz for 30 sec. Add 400 µL of Buffer MLP1 and 5 µL of RNase A (10 mg/mL). Immediately invert the tube to mix thoroughly, briefly centrifuge, and incubate in a 70 °C metal bath or water bath for 10 min. Remove the tube every 5 min and manually mix once.
2) Pretreatment Method 2: Weigh approximately 50–100 mg of freshly ground plant material powder or 15–20 mg of dried plant material powder that has been thoroughly ground in liquid nitrogen. Quickly transfer the powder into a 2 mL centrifuge tube preloaded with 400 µL of Buffer MLP1 and 5 µL of RNase A (10 mg/mL). Immediately invert the tube to mix thoroughly, briefly centrifuge, and incubate in a 70 °C metal bath or water bath for 10 min. Remove the tube every 5 min and manually mix once.
2. Centrifuge at 14,000 rpm for 4 min. Transfer 300 µL of the supernatant to a new 1.5 mL centrifuge tube.
3. Add 550 µL of Buffer MPN and 40 µL of Magnetic Beads XT. Vortex for 5 sec to mix thoroughly. Place the centrifuge tube in a thermostatic mixer at 25 °C and 1500 rpm for 5 min, or continuously invert the tube for 5 min to mix.
4. Place the centrifuge tube on a magnetic rack and let it stand for 1 min. After the magnetic beads have been completely adsorbed, discard the supernatant thoroughly while keeping the centrifuge tube fixed on the magnetic rack.
5. Remove the centrifuge tube from the magnetic rack and add 600 µL of Buffer RW1P. Vortex briefly in pulses for 1 min, or vortex for 5 sec and then place the tube in a thermostatic mixer at 25 °C and 1500 rpm for 2 min (ensure that the magnetic beads remain suspended during mixing). Then place the centrifuge tube on the magnetic rack and let it stand for 1 min. After the magnetic beads have been completely adsorbed onto the side wall of the tube, gently tilt the magnetic rack to wash down any impurities adhering to the tube cap. Discard the supernatant thoroughly while keeping the centrifuge tube fixed on the magnetic rack.
6. Remove the centrifuge tube from the magnetic rack and add 600 µL of Buffer MWP. Vortex briefly in pulses for 1 min, or vortex for 5 sec and then place the tube in a thermostatic mixer at 25 °C and 1500 rpm for 2 min (ensure that the magnetic beads remain suspended during mixing). Then place the centrifuge tube on the magnetic rack and let it stand for 1 min. After the magnetic beads have been completely adsorbed onto the side wall of the tube, gently tilt the magnetic rack to wash down any impurities adhering to the tube cap. Discard the supernatant thoroughly while keeping the centrifuge tube fixed on the magnetic rack.
7. Repeat Step 6.
8. Keep the centrifuge tube fixed on the magnetic rack and use a pipette to further remove any residual liquid from the bottom of the tube and the tube cap. Leave the tube at room temperature for 3–5 min to allow the ethanol to evaporate completely.
9. Remove the centrifuge tube from the magnetic rack and add 50–100 µL of Buffer EB. Vortex to completely resuspend the magnetic beads in the elution buffer. Place the tube in a thermostatic mixer at 65 °C and 1500 rpm for 5 min to elute the nucleic acids.
10. Transfer the centrifuge tube to the magnetic rack and let it stand for 2 min. After the magnetic beads have been completely adsorbed, carefully transfer the nucleic acid solution to a new centrifuge tube (provided by the user) and store under appropriate conditions.
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