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View additional product information for Dynabeads™ mRNA Purification Kit (for mRNA purification from total RNA preps). - FAQs (61006)
62 product FAQs found
DNA污染的原因有多种：
•DNA剪切不完全。
•在杂交步骤后，未完全去除样本裂解液。
•清洗不完全和/或洗涤缓冲液去除不完全。
•样本与磁珠的比例过高。
请查看以下可能原因：
•溶液太粘稠。
•蛋白质间相互作用导致磁珠聚集。
尝试以下建议：
•延长分离时间（将管子留在磁力架上2-5分钟）。
•向裂解液中加入DNase I（约0.01 mg/mL）。
•将结合和/或清洗缓冲液中的Tween20浓度增加至约0.05%。
•向结合和/或清洗缓冲液中加入最多至20 mM 的β-巯基乙醇。
对于小于1 kb的生物素标记DNA，我们推荐使用Dynabeads M270链霉亲和素磁珠和MyOne C1磁珠。对于大于1kb的双链DNA分子，我们推荐Dynabeads KilobaseBINDER试剂盒。KilobaseBINDER试剂包括M-280链霉亲和素偶联的Dynabeads磁珠和一种含有专利的固定活化剂的结合液，可结合较长的生物素化DNA分子以进行分离。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html)，查看关于长的生物素化DNA片段分离的更多信息。
可以，Dynabeads磁珠可用于分离单链DNA。链霉亲和素Dynabeads磁珠能够以生物素化的DNA片段为靶标，通过使双链DNA变性，从而去除非生物素化链。链霉亲和素偶联的Dynabeads磁珠不会抑制任何酶活性。因此，可以在固相上直接对磁珠结合的DNA进行下一步处理。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html)，查看关于单链DNA捕获的更多信息。
磁化率能够衡量磁珠向磁力架迁移的速度，其大小取决于铁含量和氧化铁的特性。Dynabeads磁珠的磁化率是指质量磁化率，单位可以是cgs单位/g或m^3/kg（国际单位制）。对于亚铁磁性和铁磁性物质，质量磁化率取决于磁场强度（H），这些物质的磁化强度与H不是线性关系，而是随着场强增加而趋于饱和。因此， Dynabeads磁珠的质量磁化率是在固定条件下由标准操作程序而测定的。我们产品目录中给出的质量磁化率是国际单位制。磁化率由从高斯（cgs、emu）单位向国际单位的转换，是通过“高斯系数（emu/g或cgs/g）x 4π x 10^-3”而实现的。所得单位也被称为合理化质量磁化率，与（国际单位制）无量纲磁化率单位有所区别。通常，质量磁化率可用来衡量在非均匀磁场中影响物体的力（Fz）。测定Dynabeads磁珠的质量磁化率时，首先对样本称重，然后将样本放置于已知强度的磁场中。随后，再次称重得到样本重量（F1），并与关闭磁场时样本的重量（F0）进行对比。使用下述公式计算磁化率：K x 10^–3 = [(F1-F0) x m x 0.335 x 10^6]，K表示质量为m的样本的质量磁化率。最后，将磁化率转换为国际单位制。
有多种不同的方法可以检测配体与磁珠结合，包括光密度（OD）检测、荧光标记和放射性标记。
对于OD检测，应在配体固定到磁珠上之前检测配体的OD值，并将其与包被后上清液中剩余的配体浓度进行比较。这样可以粗略检测有多少蛋白与磁珠结合。 实验方案： 1.将分光光度计设置到正确的波长。使用偶联缓冲液作为空白组。 2.检测偶联前溶液的吸光值。根据配体的加入量，可能需要进一步稀释以读取吸光值。 3.检测偶联后溶液的吸光值。也可能需要进一步稀释以读取吸光值。 4.计算偶联效率，以“蛋白质摄取量%”表示，如下所示：[(偶联前溶液的吸光值x D) – (偶联后溶液的吸光值x D)] x 100/(偶联前溶液的吸光值 x D)，D = 稀释倍数。 对于荧光标记，我们建议对配体结合量进行反向定量，即检测偶联上清液中剩余的配体量（与原始样本对比），而不是直接检测磁珠上的配体量。将标记的配体加入到磁珠中，并检测上清液中剩余多少配体（而不是结合到磁珠上的配体）。通过与开始时加入的总配体量相比，可以计算出结合到磁珠上的配体量。由于Dynabeads磁珠具有自发荧光，因此，我们不推荐直接检测与磁珠结合的配体的荧光，而是推荐这种间接方法。标记物可以是FITC/PE等。有些研究人员也成功使用了直接检测方法（采用流式细胞仪）。 在3种方法中，放射标记的灵敏度最高，但难度最大。该方法涉及到对配体的一部分进行放射性标记。在偶联前，使用示踪剂量的放射性标记的I-125，将其以一定比例与“冷”配体混合。使用闪烁（γ）计数器对磁珠进行检测，并将磁珠的cpm值与标准品对比，得到磁珠上配体的绝对量。 实验方案： 1.取出适量磁珠，并使用1 mL结合缓冲液清洗。 2.吸取适量人IgG，置于一个单独的管子中。 3.将人IgG与I-125标记的人IgG（30,000–100,000 cpm）混合。 4.使用结合缓冲液将人IgG与I-125标记的人IgG混合物稀释至100mL。 5.室温下孵育30分钟，使用闪烁计数器检测cpm值。 6.清洗磁珠（和包被层）4次，再次检测cpm值。 使用下述方程计算结合率%：（清洗后cpm值/清洗前cpm值）x100%。Dynabeads磁珠有3种尺寸：4.5 µm （M-450）、2.8 µm （M-270/M-280）和1 µm （MyOne beads）。其中最大尺寸的磁珠非常适合细胞等较大的目标，2.8 µm磁珠推荐用于蛋白质组学和分子研究，而最小的1 µm磁珠则适用于自动化处理。
一般来说，在加入配体包被磁珠时，短时间超声是减少磁珠聚集、确保磁珠获得最佳均一性的好方法。一旦目标分子结合到磁珠，就要加倍小心了，以防结合被破坏。链霉亲和素磁珠本身能够承受超声。超声5分钟是可以的，更长时间超声的影响还未被测试。关于链霉亲和素-生物素的相互作用可否被超声破坏目前也尚无相关信息。
只有未包被的环氧基或甲苯磺酰基活化的磁珠可根据需要使用70%乙醇进行清洗除菌。包被的磁珠不可灭菌。
Dynabeads磁珠是一种大小均一、无孔、超顺磁的、单分散的、高度交联的聚苯乙烯微球，整个磁珠由均匀分散的磁性材料构成。该磁性材料由磁赤铁矿(γ-Fe2O3)和磁铁矿(Fe3O4)的混合物组成。在Dynabeads磁珠M-280和M-450中，铁(Fe)分别占磁珠重量的12%和20%。Dynabeads磁珠表面覆盖有一层薄的聚苯乙烯外壳，将磁性材料包裹在内，可防止磁珠泄漏或在内部捕获配体。此外，该外壳也可避免目标分子直接接触铁，同时为每次实验提供特定的表面来吸附或偶联各种分子。
磁珠尺寸和形状均一，确保物理和化学性质稳定一致，进而提高实验结果的质量和可重复性。
Dynabeads磁珠分为3种不同尺寸：4.5 μm （M-450磁珠），2.8 μm （M-270/M-280磁珠）和1 μm （MyOne磁珠）。
从结合在Dynabeads磁珠上的mRNA生成全长cDNA是可能的。我们推荐使用一款热稳定的逆转录试剂盒，以便包含高GC二级结构的困难区域也可以被逆转录。但是，不能通过加热磁珠上的mRNA来启动反应，否则会导致mRNA的洗脱（A:T碱基对的热稳定性最差）。
我们内部尝试过使用ThermoScript逆转录酶，并以磁珠上的oligo(dT)25为引物，按照厂家说明书进行cDNA合成。当使用热稳定逆转录酶和oligo(dT)25引物进行第一链cDNA合成时，在执行推荐的高温前，有必要先在50°C孵育5分钟。这是为了启动跨越A:T杂交点的cDNA合成，从而使mRNA不会脱离磁珠。所得的cDNA共价结合到磁珠表面，结合有cDNA的磁珠可被用作多种杂交反应的模板。
在进行mRNA分离操作时，质粒会被去除。但是，如果一个polyA尾被克隆到质粒中，它将与Oligo(dT)25磁珠杂交，导致质粒被同时分离。
每毫克磁珠的结合能力为2 μg。
可以，Dynabeads Oligo(dT)25磁珠可以重复使用。请查看以下推荐条件：
重复用于相同样品：
洗脱mRNA后，使用裂解/结合缓冲液（300 μL）清洗磁珠（原始体积200 μL）1次。随后，将磁珠重新加入到样本中，进一步分离mRNA。可重复多次分离步骤，直到样本中所有mRNA均被捕获。
重复用于不同样品：
为避免样品间mRNA的残留，应通过标准磁性分离步骤使用200μL再生溶液清洗磁珠3次。其中第一次清洗时，在65°C孵育2分钟。随后，使用200μL储存缓冲液Oligo(dT)25清洗，并持续清洗至pH低于8.0。将磁珠用适量储存缓冲液Oligo(dT)25重悬。此时，磁珠已被再生，可用于后续mRNA分离。将磁珠保存于2-8°C。不可将再生的磁珠与原始磁珠储液混合。
我们提供多种用于mRNA分离的Dynabeads产品，这些产品都基于Dynabeads Oligo(dT)25磁珠。其分离原理是磁珠上共价偶联的Oligo(dT)25序列与真核生物mRNA的poly(A)尾之间的A:T碱基配对。产品如下：
•Dynabeads mRNA纯化试剂盒（货号：61006），用于从总RNA中分离mRNA。除了Dynabeads Oligo(dT)25磁珠，该试剂盒还包括结合缓冲液、洗涤缓冲液和10 mM Tris-HCl。
•Dynabeads mRNA DIRECT试剂盒（货号：61011和61012），用于从动物组织、植物组织和细胞粗提物中直接分离mRNA。除了Dynabeads Oligo(dT)25磁珠，该试剂盒还包括裂解/结合缓冲液、洗涤缓冲液、10 mM Tris-HCl、再生溶液和储存缓冲液。
•Dynabeads mRNA DIRECT 微量试剂盒（货号：61021），用于从小样本中分离mRNA。除了Dynabeads Oligo(dT)25磁珠，该试剂盒还包括裂解/结合缓冲液、洗涤缓冲液和10 mM Tris-HCl。
选择Dynabeads mRNA DIRECT试剂盒还是Dynabeads mRNA DIRECT微量试剂盒，取决于样本大小。微量试剂盒专门从小样本（例如，<4 mg的植物组织、<2 mg的动物组织、<150,000的细胞）中分离mRNA，且已被用于从少至单个细胞的微量材料中分离mRNA。Dynabeads mRNA DIRECT试剂盒可用于多达200–400 mg组织/20 x 10^6个细胞的样本。实验方案可成比例放大或缩小，以满足您的特定需求。
There are several reasons why DNA contamination may occur:
- Incomplete DNA shearing.
- Incomplete removal of sample lysate after the hybridization step.
- Insufficient washing and/or removal of wash buffers.
- The ratio of sample to beads was too high.
Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:
- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.
Try these suggestions:
- Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.
For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.
Protocol:
1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.
For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).
Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.
Protocol:
1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.
Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne beads).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
These are some references on cDNA libraries and RT-PCR:
Jakobsen KS, Haugen M, Sæbøe-Larssen S, Hollung K, Espelund M, Hornes E. Direct mRNA isolation using Magnetic Oligo (dT) Beads: A protocol for all types of cell cultures, animal and plant tissues. In Advances in Biomagnetic Separation, Ed Uhlén, M., Hornes, E., Olsvik Ø., Eaton Publishing. 1994:61-72
Raineri I, Moroni C, Senn HP. Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Research 1991;19:4010
Raineri I, Senn HP. HIV-1 promotor insertion revealed by selective detection of chimeric provirus-host gene transcripts. Nucleic Acids Res. 1992;20:6261-6266
Sharma P, Lönneborg A, Stougaard P. PCR-based construction of subtractive cDNA library using magnetic beads. BioTechniques 1993;15:610-611
Lee Y-H, Vacquier VD. Reusable cDNA libraries coupled to magnetic beads. Anal. Biochem. 1992;206:206-207
Lambert KN, Williamson VM. cDNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Research 1993;21:775-776
Aasheim H-C, Deggerdal A, Smeland EB, Hornes E. A simple subtraction method for the isolation of cell- specific genes using magnetic monodisperse polymer particles. BioTechniques 1994;16:716-721
Coche T, Dewez M, Beckers M-C. Generation of an unlimited supply of a subtracted probe using magnetic beads and PCR. Nucleic Acid Research 1994;22:1322-1323
Rodriguez IR, Chader GJ. A novel method for the isolation of tissue-specific genes. Nucleic Acids Research 1992;20:3528
Schraml P, Shipman R, Stulz P, Ludwig CU. cDNA subtraction library construction using a magnet-assisted subtraction technique (MAST). Trends in Genetics 1993;3:70-71
Wada H, Asada M, Miyazaki M, Ilda S, Mizutani S. Application of oligo (dT) Dynabeads for the molecular diagnosis of human leukemia. The John Uglestad Conference I: Magnetic separation techniques applied to cellular and molecular biology, 1991
Larsen F, Solheim J, Kristensen T, Kolstø AB, Prydz H. A tight cluster of five unrelated human genes on chromosome 16q22.1. Human Molecular Genetics 1993;2:1589-1595
The purpose of this step (heating at 65 degrees C for 5 min) is to open up secondary structures in the RNA. If you want to use the Oligo(dT)25 on the beads as primers for your cDNA synthesis and generate solid-phase cDNA, you should omit this step. Start with 50 degrees C (otherwise the mRNA will fall off the beads), then proceed to the 65 degrees C step.
It is possible to generate full-length cDNA from mRNA attached to Dynabeads magnetic beads. We recommend a thermostable reverse transcription kit, so that difficult regions with GC-rich secondary structures are accommodated. However, it is not possible to start the reaction by heating the mRNA on the beads because that will elute the mRNA (A:T base pairs are the least thermostable).
We have used ThermoScript reverse transcriptase, inhouse, with Oligo(dT)25 on the beads as primers. The cDNA synthesis was performed according to the manufacturer's instructions. When using a thermostable reverse transcriptase and the Oligo(dT)25 as primer for first-strand cDNA synthesis, an initial step of incubation at 50 degrees C for 5 min is necessary before proceeding at the recommended elevated temperature. This is to start the cDNA synthesis beyond the A:T hybridization point so that the mRNA doesn't fall off the beads. The resulting cDNA is covalently attached to the bead surface, and the beads with the attached cDNA can be used as template in multiple hybridization reactions.
Lithium chloride is included in Washing Buffer A to ensure that the mRNA remains annealed to the Oligo(dT)25 on the beads while everything else is washed away. The major advantage of using LiCl instead of other chloride salts is that LiCl does not efficiently precipitate DNA, proteins, or carbohydrates and therefore reduces the risk of contamination of the final mRNA preparation with DNA and inhibitors of cDNA synthesis, PCR etc.
LiDS is an ionic detergent, similar in function to SDS. LiDS is included in the lysis buffer is to aid in the lysis of the cells and to denature proteins, and in addition it is an effective RNase inhibitor. If you don't have LiDS in the lab, it is also possible to use SDS, but you may wish to add RNAse inhibitor as well.
Typically, a total RNA sample contains 1-3% mRNA and more than 80% rRNA, so rRNA contamination is a possibility. When mRNA is isolated using Dynabeads magnetic beads, rRNA contamination can happen due to non-specific adsorption of rRNA to the bead surface, but can also be a result of non-specific hybridization of rRNA to single-stranded mRNA. In our experience, however, the secondary structure and the well-defined sizes of rRNA can give the impression of more rRNA contamination on an agarose gel than actually might be the case. The secondary structure of rRNA also allows more EtBr intercalation, so that makes it more visible on gels than an equivalent mass of mRNA.
To reduce contaminating rRNA, we recommend a "double isolation" protocol. First, elute the mRNA from the beads in a small volume. Then wash the beads in Lysis/Binding buffer and resuspend the beads in Lysis/Binding buffer. The same beads in buffer are then combined with the eluted mRNA, and a new round of annealing and washing is performed. It is important to use the same beads, because using new beads will lead to some loss of mRNA (probably because some of the RNA will bind so tightly that it doesn't come off during elution; the original beads are already "coated" and won't participate further in nonspecific binding). Most, or all, of the rRNA will be removed by the second extraction.
Alternative/additional ways of getting rid of rRNA contamination include:
(1) increase the temperature of the washing step to 37 degrees C
(2) lower the salt concentration in the washing solution
(3) wear gloves and perform steps on ice
(4) increase volume of washing buffer used
(5) increase the number of washing steps
To purify mRNA from total RNA, we have developed the Dynabeads mRNA Purification Kit (Cat. No. 61006). Each kit is designed for 10 mRNA isolations starting from 75 µg total RNA using 200 µL beads per isolation.
It is possible to scale down these amounts for a smaller starting amount of total RNA (e.g., if using half the amount of total RNA, use half the recommended volumes of beads and buffers). At Thermo Fisher Scientific, we normally use the same protocol and the same amount of buffers and beads with a range of total RNA from 25-100 µg. An even lower amount of total RNA has been processed, following the same protocol. We recommend a 1:1 ratio between sample and binding buffer for optimal binding, and, in addition, the beads should not be diluted more than is specified in the standard protocol.
Plasmids should be removed when the mRNA isolation protocol is followed. However, if a polyA tail is cloned into the plasmid, it will hybridize with the Oligo(dT)25 beads and will result in co-isolation of the plasmid.
The capacity of Dynabeads Oligo(dT)25 magnetic beads is 2 µg/mg beads. How many transcripts can bind will depend on many factors. In a mixed pool of transcripts, the binding will be biased towards the shorter fragments. Long fragments have a tendency to interfere with bead binding sites. Long polyA tails might occupy several binding sites, as well. If the target comprises long fragments, we recommend that you use excess Dynabeads magnetic beads.
Yes, Dynabeads magnetic beads can be used for mRNA isolation from plants but there are some considerations to take into account:
Plant cells are surrounded by a very tough cell wall, which makes achieving complete cell lysis very difficult. If you are starting with plant material, cell lysis may require more manipulation than is described in the package inserts for the Dynabeads mRNA DIRECT Kit. In addition, depending on which part of the plant the mRNA is going to be isolated from, the amount of deposited starch may also interfere with mRNA isolation. Finally, you also have to consider what amount of starting material you'll have access to, what the sensitivity of the downstream methods are, and whether you want to isolate total RNA first (or go straight to the mRNA direct isolation).
Here are three references for isolation of mRNA from a small number of cells using Dynabeads mRNA DIRECT Kit:
Klein CA et al. (2002) Combined transcriptome and genome analysis of single micrometastatic cells. Nat Biotechnol 2(4):387-392.
Zhao J et al. (2001) Effect of activin A on in vitro development of rat preantral follicles and localization of activin A and activin receptor II. Biol Reprod 65(3):967-977.
Karrer EE et al. (1995) In situ isolation of mRNA from individual plant cells: Creation of cell-specific cDNA libraries. Proc Natl Acad Sci 92(9):3814-3818.
Dynabeads Oligo(dT)25 magnetic beads can be reused. Please see below for how to resue the beads depending on your sample.
Reuse: same sample
After elution of mRNA, wash the beads (original volume 200 µl) once in Lysis/Binding Buffer (300 µl). Then add the beads back to your sample for further mRNA isolation. Isolation can be repeated several times until all the mRNA is captured from the sample.
Reuse: different sample
To avoid carry-over of mRNA between different samples the beads should be washed three times in 200 µl Reconditioning Solution by standard magnetic separation. Incubate at 65 degrees C for 2 minutes at the first wash. Then wash using 200 µl Storage Buffer Oligo (dT)25 and continue carrying out washes until the pH is below 8.0. Resuspend the beads in the desired volume of Storage Buffer Oligo (dT)25. The beads are now regenerated and ready for mRNA isolation. Store the beads at 2 to 8 degrees C. Do not mix regenerated beads with the original stock suspension.
We offer several Dynabeads products for mRNA isolation, all of which are based upon the Dynabeads Oligo dT beads. The principle for isolation is A:T base paring between an oligo dT sequence covalently coupled to the beads and the PolyA tail of eukaryotic mRNA.
Dynabeads mRNA Purification Kit for the isolation of mRNA from total RNA (Cat. No. 61006: 2 mL/10 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Binding Buffer, Washing Buffer, and 10 mM Tris-HCl.
Dynabeads mRNA DIRECT kit for direct isolation of mRNA from crude extracts of animal tissue, plant tissue, and cells (Cat. No. 61011: 5 mL/20 isolations and Cat. No. 61012: 2 x 5 mL/40 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Lysis/Binding Buffer, Washing Buffers, 10 mM Tris-HCl, Reconditioning Solution, and Storage Buffer.
Dynabeads mRNA DIRECT Micro Kit for isolation of mRNA from small samples (Cat. No. 61021: 2 mL/100 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Lysis/Binding Buffer, Washing Buffers, 10 mM Tris-HCl.
Whether you choose the Dynabeads mRNA DIRECT Kit or Dynabeads mRNA DIRECT Micro Kit depends on your sample size. The micro kit is designed for mRNA isolation from small samples (e.g., <4 mg plant tissue, <2 mg animal tissue, <150,000 cells), and it has been used to isolate mRNA from as low as a single cell. Dynabeads mRNA DIRECT Kit can be used for samples up to 200-400 mg tissue/20 x 10e6 cells. The protocol can be scaled up or down to suit your specific needs.
This phenomenon can be observed after RT-PCR, but may also be observed during the washing steps in the mRNA isolation. cDNA (and the oligo dT on the beads) is negatively charged and makes the surface of the beads charged, which in repeated magnetic handling steps without detergent will give an electrostatic interaction between the beads and tube wall. The presence of salt and/or detergent (LiDS or Tween 20 detergent) in the buffer often helps to minimize these electrostatic interactions. We recommend to recondition the beads by adding up to 0.05% Tween 20 detergent (total concentration) to the sample in one washing step and leave the beads on the roller at room temperature for 5-10 min. This treatment will restore their normal appearance as well as their functionality. The detergent has to be washed away (two to three washing steps without detergent) before running any enzymatic downstream reactions (e.g., RT-PCR). Please notice that the Tween 20 detergent should be RNase free. The problem is mostly cosmetic and the performance is not changed, but sticky beads may be more difficult to work with.
The properties of Dynabeads magnetic beads are excellent for automated liquid handling. Protocols have been developed for fully automated use of Dynabeads Oligo(dT)25 magnetic beads (e.g., Biomek 2000 from Beckman Coulter and Genesis RSP from Tecan).
The isolated mRNA is highly concentrated and ideal for cloning and expression studies. The mRNA can be used directly in any downstream application in molecular biology, including northern blotting, RT-PCR, subtractive hybridization, and cDNA library construction, as documented in the published literature.
Yes. In order to assess the concentration of isolated mRNA, you need to elute the mRNA from the beads. Complete removal of the beads is necessary, as the Agilent Bioanalyzer does not produce accurate readings if any beads are present. Dynabeads solid-phase technology facilitates easy handling of your sample as well as elution of your isolated mRNA by simply applying a magnet. Therefore, it is recommended to apply the magnet a second time to remove any beads left in solution before loading the RNA chip and running the Agilent Bioanalyzer.
To use optical density measurements to determine mRNA concentration, the mRNA will first have to be eluted from the Dynabeads magnetic beads. Ensure that there are no Dynabeads magnetic beads left in the solution, as the beads will interfere with the spectrophotometrical readings.
The highly concentrated mRNA can be eluted at 65 to 80 degrees C (2 minutes) in the desired volume, typically 10 to 20 µL or down to 5 µL. If eluted in a small volume, take care to pipette off the mRNA containing supernatant as soon as the beads have migrated to the side of the tube facing the magnet. The temperature changes in such small volumes will quickly allow for re-annealing of the mRNA to the Dynabeads Oligo(dT)25 magnetic beads. The suggested elution buffer is 10 mM Tris-HCl, but DEPC-treated water may also be used. However, if the eluted mRNA should be stored, Tris-HCl is strongly recommended.
Overloading the system with excess sample material will result in a lower purity of the isolated mRNA due to nucleic acid interactions. Re-extracting the mRNA from the eluate can eliminate co-isolated rRNA. Most or all of the rRNA will be removed by the second extraction.
A general rule of thumb is not to overload the system with excess starting material, as the amount of released DNA will affect the kinetics of the nucleic acid interactions. DNA contamination in purified mRNA sample can be removed by performing a DNAse treatment if needed.
Yes, it is possible to store the lysate in the Lysis/Binding Buffer for several months at -20 degrees C. Care should be taken when thawing the sample. The sample should be thawed quickly to avoid RNase attack before the LiDS is dissolved.
Yes, a GTC based buffer (guanidinium thiocyanate) may also be used, but a dilution step is necessary to reduce the viscosity of the lysate.
Dynabeads mRNA Purification Kit supports 10 isolations from 75 µg total RNA sample.
Dynabeads mRNA DIRECT Kit supports 20 (Cat. No. 61011) or 40 (Cat. No. 61012) standard isolations.
Dynabeads mRNA DIRECT Micro Kit supports 100 isolations from small samples.
Dynabeads-based mRNA isolation involves fewer handling steps, thereby minimizing loss and sample degradation. Using the biomagnetic separation technology, centrifugations and ethanol precipitations are eliminated. Phenol chloroform extractions are no longer necessary. In addition, there is no need for a total RNA preparation step, as mRNA can be isolated directly from the crude starting sample. Buffer changes and further downstream handling of the mRNA are easily performed with a magnet.
Which product/protocol to choose depends on the sample you have:
Dynabeads mRNA Purification Kit is designed for mRNA isolation from total RNA samples. This kit contains a Binding Buffer for specific binding of mRNA to the oligo dT sequence on the beads.
Dynabeads mRNA DIRECT Kit is designed for direct isolation of mRNA from crude lysates of animal and plant cells and tissues. Sample size is typically 2-400 mg tissue or 0.1-20 x 10e6 cells. This kit contains a Lysis/Binding Buffer for both lysis and specific binding of mRNA to oligo dT on the beads.
Dynabeads mRNA DIRECT Micro Kit is designed for direct isolation of mRNA from very small tissue and cell samples. The principle is similar to Dynabeads mRNA DIRECT Kit, but the protocol is optimized for mRNA isolation from less than 2-4 mg tissue or less than 150,000 cells. This kit has been used for mRNA isolation from down to one single cell. (When mRNA isolation is performed from small samples, we recommend to do RT-PCR directly on the beads without elution).
mRNA isolation is based upon hybridization of the poly A+ tailed RNA (mRNA) to the oligo(dT) covalently coupled to Dynabeads magnetic beads. The Binding Buffer and Lysis/Binding Buffer in mRNA isolation kits are optimized for this hybridization.
The M stands for magnetic. M-280 refers to hydrophobic 2.8 micron beads, while M-270 refers to hydrophilic 2.8 micron beads. MyOne refers to 1 micron beads.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, volumes less than 10 µL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads with a lot of bound target in a small elution volume, your elution may not be very efficient. Typically, 15-100 µL of beads may be eluted in 30 µL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30-40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads magnetic beads.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Using Dynabeads magnetic beads for protein isolation provides several advantages:
-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.
-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.
Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.
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Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.
High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.
However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.