Search
Search
View additional product information for Dynabeads™ mRNA DIRECT™ Micro Purification Kit - FAQs (61021)
49 product FAQs found
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.
Both genomic DNA and mRNA can be isolated from the same sample, but we recommend that you isolate the DNA first using the Dynabeads DNA DIRECT Universal Kit in order to avoid large DNA fragments binding to the Oligo(dT)25 beads during the mRNA isolation procedure.
After genomic DNA is isolated, you can use the Dynabeads mRNA DIRECT Kit or Dynabeads mRNA DIRECT Micro Kit to isolate the mRNA from the same lysate (just add the Dynabeads Oligo (dT)25 magnetic beads to the lysate, don't use the lysis/binding buffers in the kit, and follow the remainder of the protocol).
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.
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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.