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Oligo(dT)18 (NH₂-COOH) 磁珠
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为什么选择此级别
BioReagent,分子生物学级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
阿拉丁 Oligo(dT)₁₈ (NH₂-COOH) 磁珠，以羧基磁珠为基质，通过酰胺共价键固定 5' 氨基修饰 Oligo(dT)₁₈。磁珠粒径约 1 μm，分散性优异、磁含量高，磁响应迅速，且 Oligo(dT) 载量适中可控。 该产品依托表面 Oligo(dT) 与 mRNA Poly (A) 尾的特异性互补配对，可从真核总 RNA、动植物组织及细胞样本中高效捕获完整 mRNA，获得高纯度目的产物。所得 mRNA 适用于 RT-PCR、cDNA 文库构建、Northern Blot、引物延伸、基因表达分析等各类下游分子生物学实验。
产品信息
| 产品信息 | O1539201 |
|---|---|
| Oligo(dT)载量 | ＞500 pmol / mg 磁珠 |
| 浓度 | 5 mg/mL |
| 粒径 | 约 1.0 μm |
| 磁含量 | 约 35%-45% |
| 保存溶液 | TE，含 0.1%BSA 和 0.01%P300 作为防腐剂 |
| 保存条件 | 密封，2-8°C保存，禁止冷冻，使用前请充分混匀 |
| 包装 | 塑料瓶 |
操作步骤
1、准备缓冲溶液和材料
1.1 缓冲液配制：用户可根据需要自行调整缓冲液配方（所有试剂需要使用 DEPC 处理的水配制）
| 溶液名称 | 溶液配方 |
|---|---|
| 结合缓冲液 | 100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1% LiDS; 5 mM dithiothreitol |
| 洗涤缓冲液 A | 10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA; 0.1% LiDS |
| 洗涤缓冲液 B | 10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA |
| 洗脱液 | 10 mM Tris-HCl pH 7.5 |
1.2 RNase-free 的 1.5 mL 离心管；
1.3 用于 1.5 mL 离心管的磁力架；
1.4 焦碳酸二乙酯（DEPC）水：向纯化水中加入终浓度 0.1%(V/V) 的 DEPC，混匀后放置过夜，高压灭菌。
1.5 水浴锅；
1.6 涡旋振荡器
1.7 旋转混合仪或摇床
2、清洗 Oligo(dT)磁珠
2.1 彻底重悬试剂瓶中的 Oligo(dT)磁珠（摇床振荡 10 min 或手动摇晃至充分混匀）。
2.2 将所需体积的 Oligo(dT)磁珠转移到离心管中，加入结合缓冲液，使磁珠浓度为 0.4 mg/mL。
2.3 将离心管置于磁力架上 1 min，弃上清。
吸弃上清时，尽量吸净管盖及管底残液，请勿吸入磁珠。
2.4 将离心管从磁力架上取下，加入与初始体积相同体积的结合缓冲液，留作下一步备用。
3、从总 RNA 中纯化 mRNA
3.1 将使用结合缓冲液洗涤并重悬的磁珠充分混匀后，取 50 μL 至离心管中。
3.2 样品准备：用 DEPC 水将 10 μg 总 RNA 样品的体积调整至 50 μL，加入含 50 μL 磁珠的离心管中，移液器轻柔混匀。
3.3 样品置于 PCR 仪，65°C 5 min，25°C 5 min，4°C hold。
3.4 磁分离，去上清，使用 200 μL 洗涤缓冲液 A 洗涤结合 mRNA 的磁珠，移液器轻柔混匀，磁分离，去上清。
注：完成清洗时务必清除所有洗涤缓冲液。
3.5 加入 50 μL 洗脱液，移液器轻柔混匀，将样品置于 PCR 仪，80°C 2 min，25°C hold。
3.6 加入 50 μL 结合液，移液器轻柔混匀，室温放置 5 min；再磁分离，去上清。
3.7 加入 200 μL 洗涤缓冲液 A，移液器轻柔混匀，磁分离，去上清。
注：完成清洗时务必清除所有洗涤缓冲液。
3.8 加入 200 μL 洗涤缓冲液 B，移液器轻柔混匀，磁分离，去上清。重复此步骤两次。
注：完成清洗时务必清除所有洗涤缓冲液。
3.9 从磁力架上取下离心管，加入 10 μL DEPC 水或洗脱液，重悬磁珠，80°C 2 min。
3.10 迅速使用磁力架收集上清，并将上清液转移至无 RNase 的离心管中，进行下游实验。
注意事项
1、尽量减少 RNA 降解；使用 RNA 时，请始终佩戴手套以尽量减少 RNase 污染；所有用于 mRNA 提取的 buffer 和耗材都应该是 RNase-free。
2、为减少磁珠损失，每次磁分离时间应不少于 1 min。
3、在洗涤过程中彻底重新悬磁珠/mRNA 复合物，并在每一步中完全去除洗涤缓冲液，以防止 LiDS 和其他盐携带到下游反应中。LiDS 是酶促反应的强抑制剂。
4、如果纯化后的 rRNA 残留量对下游应用过高，建议用新的磁珠进行第二轮纯化 mRNA。
5、本产品仅供研究使用。
Aladdin Oligo(dT)₁₈ (NH₂-COOH) Magnetic Beads use carboxyl magnetic beads as the substrate. 5'-amino-modified Oligo(dT)₁₈ is immobilized via covalent amide bonds. The beads have a particle size of approximately 1 μm, excellent dispersity, high magnetic content and rapid magnetic response, with moderate and controllable Oligo(dT) loading capacity. Based on the specific complementary base pairing between surface Oligo(dT) and the poly(A) tail of mRNA, this product efficiently captures intact mRNA from eukaryotic total RNA, animal and plant tissues as well as cell samples to obtain high-purity target products. The resulting mRNA is suitable for various downstream molecular biology experiments, including RT-PCR, cDNA library construction, Northern blot, primer extension, gene expression analysis, etc.
Product Information
| Product Information | O1539201 |
|---|---|
| Oligo(dT) Loading Capacity | >500 pmol / mg beads |
| Concentration | 5 mg/mL |
| Particle Size | Approximately 1.0 μm |
| Magnetic Content | Approximately 35%-45% |
| Storage Solution | TE, containing 0.1% BSA and 0.01% P300 as preservatives |
| Storage Conditions | Store sealed at 2-8°C. Do not freeze. Mix thoroughly before use |
| Packaging | Plastic bottle |
Procedure
1、Preparation of Buffers and Materials
1.1 Buffer preparation: Users may adjust the buffer formulations according to their needs (all reagents must be prepared using DEPC-treated water).
| Solution Name | Solution Formulation |
|---|---|
| Binding Buffer | 100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1% LiDS; 5 mM dithiothreitol |
| Washing Buffer A | 10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA; 0.1% LiDS |
| Washing Buffer B | 10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA |
| Elution Buffer | 10 mM Tris-HCl pH 7.5 |
Note: Equilibrate all reagents to room temperature before use. If precipitation occurs, preheat at 37°C for 10 min.
1.2 RNase-free 1.5 mL centrifuge tubes;
1.3 Magnetic rack for 1.5 mL centrifuge tubes;
1.4 Diethyl pyrocarbonate (DEPC) water: Add DEPC to purified water to a final concentration of 0.1% (V/V), mix thoroughly, leave overnight, and autoclave.
1.5 Water bath;
1.6 Vortex mixer
1.7 Rotating mixer or shaker
2、Washing Oligo(dT) Magnetic Beads
2.1 Thoroughly resuspend the Oligo(dT) magnetic beads in the reagent bottle (shake on a shaker for 10 min or shake manually until fully mixed).
2.2 Transfer the required volume of Oligo(dT) magnetic beads into a centrifuge tube and add binding buffer to obtain a bead concentration of 0.4 mg/mL.
2.3 Place the centrifuge tube on a magnetic rack for 1 min and discard the supernatant.
When aspirating the supernatant, remove as much residual liquid as possible from the tube cap and bottom. Do not aspirate the magnetic beads.
2.4 Remove the centrifuge tube from the magnetic rack and add a volume of binding buffer equal to the initial volume for use in the next step.
3、Purification of mRNA from Total RNA
3.1 After thoroughly mixing the magnetic beads washed and resuspended in binding buffer, transfer 50 μL into a centrifuge tube.
3.2 Sample preparation: Adjust the volume of a 10 μg total RNA sample to 50 μL with DEPC water, add it to the centrifuge tube containing 50 μL of magnetic beads, and mix gently by pipetting.
3.3 Place the sample in a PCR instrument at 65°C for 5 min, 25°C for 5 min, and 4°C hold.
3.4 Perform magnetic separation and discard the supernatant. Wash the magnetic beads bound to mRNA with 200 μL of Washing Buffer A, mix gently by pipetting, perform magnetic separation, and discard the supernatant.
Note: Be sure to remove all washing buffer after washing.
3.5 Add 50 μL of elution buffer, mix gently by pipetting, and place the sample in a PCR instrument at 80°C for 2 min, followed by 25°C hold.
3.6 Add 50 μL of binding solution, mix gently by pipetting, and incubate at room temperature for 5 min; then perform magnetic separation and discard the supernatant.
3.7 Add 200 μL of Washing Buffer A, mix gently by pipetting, perform magnetic separation, and discard the supernatant.
Note: Be sure to remove all washing buffer after washing.
3.8 Add 200 μL of Washing Buffer B, mix gently by pipetting, perform magnetic separation, and discard the supernatant. Repeat this step twice.
Note: Be sure to remove all washing buffer after washing.
3.9 Remove the centrifuge tube from the magnetic rack, add 10 μL of DEPC water or elution buffer, resuspend the magnetic beads, and incubate at 80°C for 2 min.
3.10 Quickly collect the supernatant using the magnetic rack and transfer it to an RNase-free centrifuge tube for downstream experiments.
Precautions
1、Minimize RNA degradation. Always wear gloves when handling RNA to minimize RNase contamination. All buffers and consumables used for mRNA extraction should be RNase-free.
2、To minimize magnetic bead loss, each magnetic separation should last at least 1 min.
3、Thoroughly resuspend the magnetic bead/mRNA complexes during washing and completely remove the washing buffer at each step to prevent LiDS and other salts from being carried over into downstream reactions. LiDS is a strong inhibitor of enzymatic reactions.
4、If the residual rRNA level after purification is too high for downstream applications, it is recommended to perform a second round of mRNA purification using fresh magnetic beads.
5、This product is for research use only.
规格
规格或纯度
BioReagent, 分子生物学级, 5 mg/mL
稳定性与储存
Store at 2-8℃ long term (24 months).Do not freeze.Avoid freeze/thaw cycle.
英文名称
Oligo(dT)18 (NH₂-COOH) Magnetic Beads
储存条件
2-8°C储存
运输条件
低温运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
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