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Dynabeads™ Oligo(dT)25, 2 mL - FAQs

Dynabeads™ Oligo(dT)25, 2 mL - FAQs

查看更多产品信息 Dynabeads™ Oligo(dT)25 - FAQs (61005, 61002)

57 个常见问题解答

我在使用Dynabeads磁珠分离mRNA时出现了DNA污染，为什么会出现这种情况？

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。

我在使用Dynabeads磁珠分离mRNA时出现了rRNA污染，该如何处理？

通过从洗脱液中再次提取mRNA，可以有效清除核糖体RNA。可重复利用初始分离时所使用的相同Dynabeads Oligo(dT)25磁珠。使用洗涤缓冲液B清洗磁珠2次。以4倍体积的裂解/结合缓冲液稀释洗脱mRNA，随后加入磁珠。在室温下孵育并混匀3-5分钟，随后继续执行直接mRNA分离实验方案。

分离的mRNA可以在Dynabeads Oligo(dT)25磁珠上保留多久？

我们推荐立即将Dynabeads磁珠-mRNA复合物或洗脱的mRNA用于RT-PCR的cDNA合成或其他下游应用。如果需要保存，我们推荐使用10 mM Tris-HCl缓冲液（pH 7.5）从磁珠上洗脱mRNA并冷冻保存（-80°C）。所有设备和样本都必须是无RNase的。

我的Dynabeads磁珠不能很好地吸附到磁力架上，对此你们有什么建议吗？

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。

在实时PCR中是否可以使用Dynabeads Oligo(dT)25磁珠？

Dynabeads磁珠可与TaqMan实时PCR反应和非毛细管实时PCR仪器兼容。但是，Dynabeads磁珠具有低水平的自发荧光，在一定程度上会增强PCR反应的荧光信号强度。这可通过在仪器中设置Dynabeads磁珠和水的背景而进行补偿。随后，在所有包含Dynabeads磁珠的实时PCR实验中，可从样本信号强度中减去背景信号强度。或者，在使用标准曲线法分析时，可在用于构建标准曲线的每个样本中均加入适量Dynabeads磁珠。

我想要分离较长的双链DNA片段，你们有什么产品可以推荐？

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。

我能否使用Dynabeads磁珠分离单链DNA模板？

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。

什么是磁化率？

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。

我如何确定Dynabeads磁珠的偶联效率？

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磁珠有哪些尺寸？

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。

Dynabeads磁珠可使用超声处理么？

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。

Dynabeads磁珠能否灭菌？

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。

什么是Dynabeads磁珠？

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）。

I am getting DNA contamination after mRNA isolation using Dynabeads magnetic beads. Why is this?

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.

I am getting rRNA contamination after mRNA isolation using Dynabeads magnetic beads. What should I do?

Ribosomal RNA is effectively eliminated by reextracting the mRNA from the eluate. Reuse the same Dynabeads Oligo(dT)25 beads that were used for the original isolation. Wash the beads twice in Washing Buffer B. Dilute the eluted mRNA with 4 times its volume of Lysis/Binding Buffer, then add the beads. Incubate with mixing at room temperature for 3-5 minutes, then continue with the Direct mRNA Isolation Protocol.

How long can I leave the isolated mRNA on Dynabeads Oligo(dT)25 magnetic beads?

We recommend immediate use of Dynabeads magnetic beads-mRNA complex or eluted mRNA for cDNA synthesis, in RT-PCR, or for other downstream applications. If storage is needed, we recommend you elute the mRNA from the beads using 10 mM Tris-HCl buffer (pH 7.5) and freeze it(-80°C). It is very important that all equipment and samples are RNase free.

My Dynabeads magnetic beads are not pelleting well with the magnet. Do you have any suggestions for me?

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.

Can I use Dynabeads Oligo(dT)25 magnetic beads in real-time PCR?

Dynabeads magnetic beads are compatible with TaqMan real-time PCR chemistry and non-capillary real-time PRC instruments. However, Dynabeads magnetic beads exhibit a low level of autofluorescence that can increase the intensity of the fluorescent signal to some degree. This can be compensated for by using a Dynabeads magnetic beads and water background in the instrument. Then background signal intensity can be subtracted from the sample signal intensity in all subsequent real-time PCR experiments containing Dynabeads magnetic beads. Alternatively, when using the standard curve method of analysis, an appropriate amount of Dynabeads magnetic beads can be added to each sample used to construct the standard curve.

I have a long double-stranded DNA fragment I would like to isolate. What product do you recommend?

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.

Can I use Dynabeads magnetic beads to isolate single-stranded DNA templates?

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.

What is the magnetic susceptibility for Dynabeads magnetic beads?

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.

How can I determine coupling efficiency of Dynabeads magnetic beads?

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.

What sizes do you offer for the Dynabeads magnetic beads?

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.

Can Dynabeads magnetic beads be sonicated?

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.

Can Dynabeads magnetic beads be sterilized?

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.

What are Dynabeads magnetic beads?

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.

After isolation of mRNA using Dynabeads Oligo(dT)25 and before doing reverse transcription, should I incubate the beads with bound mRNA attached to the primer, at 65 degrees C for 5 min as suggested in my reverse transcription protocol or should I just move on to cDNA synthesis (with incubation at 50 degrees C and then 65 degrees C?

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.

Can I use the Dynabeads Oligo(dT)25 products to isolate RNA from bacteria?

Prokaryotic mRNA does not (usually) contain a poly A+ tail, which means that Dynabeads Oligo(dT)25 magnetic beads cannot be used directly in such isolations. On the other hand the following article describes a method to polyadenylate bacterial polysomes (the assemblage of mRNA, ribosomes, and growing peptide chain during translation), which can then be isolated using Dynabeads Oligo(dT)25 magnetic beads:
Amara RR et al., (1997) Specific polyadenylation and purification of total messenger RNA from E. coli, Nucleic Acid Research, vol 25 (17), 3465-3470.
It is also possible to isolate specific RNA by specific capture using Dynabeads Streptavidin magnetic beads with biotinylated probe.

The beads seem sticky, what can I do to avoid this?

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.

If the Dynabeads Oligo(dT)25 magnetic beads dries, will they still be functional?

Do not leave the beads unsuspended for a long period of time. Vials containing Dynabeads magnetic beads should be stored upright to ensure that the beads are covered with buffer. Drying of Dynabeads magnetic beads may reduce their efficiency. If Dynabeads magnetic beads do become dried, they should be resuspended by keeping the vial in motion on a roller for up to 12 hours (4 degrees C). This may restore the functionality of the Dynabeads magnetic beads.

Can I automate the mRNA isolation protocol?

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).

Can the Dynabeads Oligo(dT)25 magnetic beads be used to create cDNA libraries?

Yes. Using Dynabeads Oligo(dT)25 magnetic beads, a solid-phase reusable cDNA library can be created directly on the bead surface, without elution of the mRNA. The bead-bound oligo(dT) is then used as primer for first-strand cDNA synthesis.

For which applications can the isolated mRNA be used?

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.

Can I use Agilent Bioanalyzer to determine amount of isolated mRNA?

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.

Can I measure the amount of isolated mRNA by an OD reading?

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.

How should I store the mRNA isolated with Dynabeads Oligo(dT)25 magnetic beads?

Generally, we don't recommend storing mRNA because it is not stable. Therefore, we recommend immediate use of Dynabeads-mRNA complex or eluted mRNA in RT-PCR (cDNA synthesis) or other downstream application. If storage is needed, we recommend to elute the mRNA from the beads, using a 10 mM Tris-HCl buffer (pH 7.5) and freeze (-80 degrees C). It is very important that all equipment and samples are RNase free. We do not recommend that you store the mRNA on the beads. However, it is possible to store the complex for a couple of hours in Tris-HCl buffer at 4 degrees C.

In what volumes can the isolated mRNA be eluted?

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.

How can rRNA contamination be avoided in mRNA isolation?

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.

How can DNA contamination be avoided in mRNA isolation?

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.

After homogenizing the tissue, can the lysate be stored in Lysis/Binding Buffer, when using the Dynabeads mRNA isolation kits ?

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.

Can GTC based buffer be used instead of the Lysis/Binding Buffer recommended in Dynabeads mRNA isolation protocols?

Yes, a GTC based buffer (guanidinium thiocyanate) may also be used, but a dilution step is necessary to reduce the viscosity of the lysate.

How many mRNA isolations can be performed using each Dynabeads mRNA isolation kit?

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.

In what way do the Dynabeads mRNA isolation protocols differ from the traditional techniques?

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 of the three Dynabeads mRNA isolation products/protocols should I choose?

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).

What is the principle for mRNA isolation using Dynabeads Oligo(dT)25 magnetic beads?

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.

What is the expected yield of mRNA from total RNA?

mRNA yield is usually 1 to 3% of the total RNA. For example, if you begin with 100 µg of total RNA, you should expect a yield of 1 to 3 µg of mRNA.

What is the detection limit when using Dynabeads magnetic beads for immunoprecipitation (IP)?

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.

What is the elution volume when using Dynabeads magnetic beads for immunoprecipitation (IP)?

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.

How can I quantify the amount of antibody bound to Dynabeads magnetic beads?

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.

How long should I incubate my antibody with the lysates?

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.

When should I covalently bind the antibody to the Dynabeads surface?

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.

What are the general advantages of using Dynabeads magnetic beads for protein isolation?

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.

Are Dynabeads magnetic beads compatible with dithionite, DTT, and EDTA?

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.

Are Dynabeads magnetic beads compatible with Urea?

Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Are Dynabeads magnetic beads compatible with centrifugation?

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.

What are the benefits of using magnetic beads in immunoprecipitation (IP) experiments?

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.