Dynabeads® Nucleic Acid Purification Support—Getting Started
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Properties of 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).
This is a challenging property to measure. The reason is that the Dynabeads® magnetic beads have a content of 17–37% magnetic iron oxide so the magnetic beads have a reasonable magnetic separation time, and the density of the iron oxide is about 4.9. Dynabeads® magnetic beads are composite materials, being a mix of polymers and iron oxide, and there are very few polymers that have a density below 1. The sedimentation rate depends on the square of the bead diameter, so the sedimentation of a 1 µm bead is much slower than 2.8 µm bead. The effect of going down in size is to some extent counteracted by the fact that smaller beads need to have a higher content of iron oxide to be used in magnetic separation. Typically our M-280 beads have a density of 1.4 and our 1 µm beads a density of 1.8. Increasing the density of the water phase should also decrease the sedimentation rate.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
In general, short sonication is a good way to reduce aggregation of the beads and insure 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.
Centrifugation of raw material beads has been tested at 6,000 rpm. Higher speeds have not been tried. However, Dynabeads® magnetic beads 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.
This is dependent on coating or the biotinylated molecule properties. Our recommendation is that this should be tested to find optimum conditions for the specific assay.
Two important factors for all solvents are concentration and incubation time. The beads themselves are compatible with a number of solvents that leave the iron oxide precipitates untouched, such as:
Ethanol (70% is actually used to sterilize the beads and for washings), methanol, acetone, isopropanol, diglyme, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, and tetrahydrofuran (THF). THF is similar to diglyme and should work fine with Dynabeads® magnetic beads.
Yes, they are compatible with 6–8 M urea.
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.
Dynabeads® Magnetic Beads - General
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.
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.
- Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
- 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.
- Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
- 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.
- Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
- Pipette out desired amount of human IgG in a separate tube.
- Mix the human IgG with I-125-labeled human IgG (30,000–100,000 cpm).
- Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
- Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
- 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%.
M stands for magnetic and 280 and 270 are an indication of the diameter of the beads, both 2.8 µm. M-280 refers to hydrophobic 2.8 µm beads while M-270 refers to hydrophilic 2.8 µm beads. MyOne™ refers to 1 µm 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.
All Dynabeads® magnetic beads are either coated with a ligand or are surface activated, meaning they have a chemical functionality on their surface.
( 1) The most inert beads are Dynabeads® M-270 magnetic beads and Dynabeads® MyOne™ Carboxylic Acid magnetic beads. They need activation (with EDC) to react covalently with molecules. One problem is that these beads may form clumps or aggregate and can be difficult to handle at neutral pH (they are easier to handle at lower pH, about 5-6).
(2) Dynabeads® M-270 Epoxy magnetic beads can be inactivated by incubation in aqueous solutions (the reason they are shipped freeze-dried), and these beads will become inert due to hydrolysis when they come in contact with water (the required incubation time is unknown, but fairly long). (Please note that Dynabeads® M-450 Epoxy magnetic beads is more stable, as it is based on a more hydrophobic particle). Incubation in Tris buffer may also effectively block the epoxy groups (at least a 4 hours incubation at room temperature). Using Tris will add amine groups, and to reduce this effect, it may be better to incubate the coated beads overnight in ethanolamine (0.1 M at 60 degrees C for 1 h or 37 degrees C overnight). This will also inactivate epoxy groups. In addition, you can accelerate the hydrolysis by incubation in water with a high concentration of NaOH (0.5 M at 60 degrees C for 1 h or 37 degrees C overnight). There are also chemical groups on the bead surface other than the remaining epoxy groups that can cause adsorption of molecules.
(3) Smaller beads (because they are more unstable) act as more inert beads.
(4) Tosylactivated beads (both Dynabeads® M-280 Tosylactivated magnetic beads and Dynabeads® M-450 Tosylactivated magnetic beads) may be inactivated by blocking the active groups with Tris alone. But the first two approaches are likely to work better.
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 for more information in regards to single-stranded DNA capture.
For biotin-labled DNA that is less than 1 kb, we recommend you use Dynabeads® M270 Streptavidin and MyOne™ C1 magnetic beads. We recommend our Dynabeads® KilobaseBINDER™ Kit, 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 for more information in regards to long biotinylated DNA fragment isolation.
The DynaMag™-2 Magnet can hold 16 standard 1.5 mL or 2 mL microcentrifuge tubes, while the DynaMag™-Spin Magnet holds up to 6 standard 1.5 mL microcentrifuge tubes.
Spray and/or wipe the DynaMag™ unit with one of the following cleaning agents:
- 70% isopropyl alcohol
- 1% sodium hypochlorite solution (bleach)
- 0.1 N HCl solution
Do not submerge the unit in aqueous solutions and avoid prolonged exposure to water or solutions. Do not autoclave or heat the magnet above 70°C.
1 Tesla is equivalent to 10,000 Gauss.
The DynaMag™-2 Magnet is 3,500–3,700 minimum Gauss level and the DynaMag™-Spin is 3,000–3,500 minimum Gauss level.
Streptavidin-coupled Dynabeads® Magnetic Beads
One milligram of Dynabeads® M-280 Streptavidin magnetic beads typically binds 650–900 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 μg of biotinylated antibody, 10 μg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
One milligram of Dynabeads® M-270 Streptavidin magnetic beads typically binds more than 950 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 μg of biotinylated antibody, 10 μg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
One milligram of Dynabeads® MyOne™ Streptavidin C1 magnetic beads typically binds more than 2,500 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 μg of biotinylated antibody, 20 μg of biotinylated double-stranded DNA, or 500 pmol of biotinylated single-stranded oligonucleotides.
One milligram of Dynabeads® MyOne™ Streptavidin T1 magnetic beads typically binds 1,100–1,700 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 μg of biotinylated antibody, 20 μg of biotinylated double-stranded DNA, or 400 pmol of biotinylated single-stranded oligonucleotides.
In direct capture, the biotinylated probe/ligand is first coupled to the Dynabeads® magnetic beads followed by addition of your sample. In indirect capture, the biotinylated probe/ligand is first added to the sample followed by addition of your Dynabeads® magnetic beads. Precoupled ligand for direct capture allows you to reuse the Dynabeads® magnetic beads, while an indirect approach can be beneficial when the concentration of your target is low, specific affinity is weak, or the binding kinetics is slow. Please see the following link for a diagram of the capture.
All biotin reagents should contain a spacer arm, at least a 6-carbon linker, to reduce steric hindrance. This is because the bicyclic ring of biotin goes deep into the biotin binding cleft in streptavidin. A 6 carbon arm is the minimum length between biotin and the first base of sequence that is required to reduce the steric hindrance effect. The longer this distance is, the less the steric hindrance. A 6-carbon linker is standard linker size from most companies and should be enough for most applications. We recommend specific biotinylation at the 5'-end of the probe.
The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per milligram Dynabeads® M-280 Streptavidin magnetic beads.
Streptavidin is a protein made up of four identical subunits, each containing a high affinity binding site for biotin (KD = 10–15 M). Streptavidin has the same biotin binding properties as avidin, but less nonspecific binding is observed. After immobilization on the beads, there are 2–3 binding sites free for interaction with biotin.
Our Dynabeads Streptavidin magnetic beads can be used directly in PCR or real-time PCR. However, you would have to empirically optimize the amount of beads to be used per volume of reaction.
You can assay the supernatant for unbound nucleic acid to determine the amount of nucleic acid bound to the Dynabeads® Streptavidin beads. The concentration of nucleic acids can be checked by measuring the OD or by running them on a gel. Alternatively, the nucleic acids can be labeled radioactively and the concentration measured directly on the beads, or fluorescently and measured in the supernatant.
The streptavidin-biotin interaction is the strongest known noncovalent biological interaction between a protein and other molecule. The bond formation between biotin and streptavidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment, requiring a specific form and a normally gentle way to dissociate biotin from streptavidin, very harsh methods are often required to dissociate the biotin from streptavidin, which will denature the streptavidin. A couple of these methods are discussed below.
Biotinylated nucleic acids: To dissociate biotinylated nucleic acids from Dynabeads® Streptavidin magnetic beads, incubate the beads in 95% formamide + 10 mM EDTA, pH 8.2, for 5 minutes at 65°C or for 2 minutes at 90°C. Pull the beads to the tube wall with the magnet and remove the supernatant containing the biotinylated nucleic acid from the tube. Holmberg et al. (Electrophoresis 26, 501-510, 2005), report release of biotinylated DNA from streptavidin beads after short incubation in deionized H2O (though we have not tested this).
Biotinylated proteins: For biotinylated proteins, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 min.
Which product to choose depends on the properties of your sample, the buffers and solutions applied, as well as the downstream application. In general, all Dynabeads® Streptavidin beads can be used in applications involving biotinylated ligands; however, some beads may perform better than others in particular applications due to their characteristics. Dynabeads® M-280 Streptavidin beads and Dynabeads® MyOne™ Streptavidin T1 beads are commonly used for protein and nucleic acid applications. Dynabeads® M-270 Streptavidin beads and MyOne™ Streptavidin C1 beads are preferred for nucleic acid diagnostics, specifically with samples that have a high concentration of chaotropic salts, immunoassays involving small biotinylated antigens, and in applications that are not compatible with BSA, as these beads are not blocked with BSA. Dynabeads® MyOne™ Streptavidin beads offer increased binding capacity and slower sedimentation rate, making them ideal for automated applications and when larger amounts of a biotinylated compound or its specific target need to be isolated. Please refer to the selection guide here.
The capacity is dependent on the fragment size due to steric hindrance. For example, twice as many 500 bp fragments will bind to Dynabeads® M-280 Streptavidin magnetic beads than a 1,000 bp fragment. Long DNA fragments will occupy more space around the beads and make it more difficult to "find" the streptavidin on the beads. Smaller fragments will access the streptavidin more easily. For DNA fragments greater than 2 kb, the Dynabeads® kilobaseBINDER™ Kit is recommended. This kit contains Dynabeads® M-280 Streptavidin magnetic beads and a special binding solution that enhances immobilization of long (greater than 2 kb) biotinylated DNA fragments. For DNA fragments greater than 1-2 kb, the Dynabeads® kilobaseBINDER™ Kit is recommended, as the binding solution will enhance binding capacity. The binding solution will linearize the DNA so that it stretches out and the bases stack in a rigid structure (it will not work for shorter fragments such as plasmids or circular nucleic acids).
The salt concentration influences the efficiency of binding of biotinylated nucleic acids to the streptavidin-coupled Dynabeads® magnetic beads. Optimal binding conditions for biotinylated DNA fragments (up to 1 kb) are achieved at 1 M NaCl (final concentration), 25 degrees C, and 15 min incubation time. Longer DNA fragments should be immobilized overnight. Biotinylated antibodies should be immobilized in PBS buffer, pH 7.4, supplemented with 0.1% BSA. Ensure that your sample does not contain excess free biotin, as the free biotin will bind Dynabeads® Streptavidin Beads much more rapidly than larger molecules. Biotinylated oligonucleotides should be recovered by reverse phase HPLC or FPLC to avoid free biotin from being present in the sample. Titration is performed to optimize the quantity of beads used for each individual application, since both the size of the specific molecule to be immobilized and the biotinylation procedures will affect the binding capacity of the beads.
Dynabeads® Streptavidin beads are not supplied in RNase-free solution. For RNA manipulations, the beads should be washed twice for 1–3 minutes in a DEPC-treated 0.1 M NaOH/0.05 M NaCl solution. DEPC is very toxic but will get rid of RNases. After washing, the beads can be resuspended in a DEPC-treated 0.1 M NaCl solution.
DEPC-treated means: add 0.1% DEPC, mix, incubate for 1 hour at room temperature and then autoclave the DEPC-treated solution to destroy the DEPC).
Two DNA strands can be separated by treating either with alkali or high temperature. Using alkali, you elute off the non-biotinylated strand with 0.1 M NaOH. This treatment should normally not have any effect on the beads.
- Wash the Dynabeads®/DNA complex once in 2X binding and washing buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl) prior to NaOH treatment and remove the supernatant. The high salt concentration will help to reduce the charge and hence minimize nonspecific binding.
- Add freshly made (important) 0.1 M NaOH to the Dynabeads®/DNA complex and incubate at room temperature for 2–3 minutes (maximum 5 minutes) with rotation. Remove the supernatant containing the non-biotinylated strand.
- Wash the Dynabeads®/DNA complex once more with 0.1 M NaOH and remove the supernatant. Most of the DNA will come off during the first elution.
Heating at 95°C for 5 minutes in water is an alternative to the alkali treatment.
To prevent reannealing, separation of the DNA from the beads must be done quickly after alkali or high temperature treatment, preferably on ice. Heating will cause some percentage (approx. 7% in water) of biotinylated DNA to be dissociated from streptavidin. Therefore, we usually recommend alkali treatment.
mRNA isolation is based upon hybridization of 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.
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 pairing between an oligo(dT)25 sequence covalently coupled to the beads and the poly(A) tail of eukaryotic mRNA. These are the products:
- Dynabeads® mRNA Purification Kit for the isolation of mRNA from total RNA (Cat. No. 61006). 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. Nos. 61011, 61012). 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). In addition to Dynabeads® Oligo(dT)25 magnetic beads, the kit contains lysis/binding buffer, washing buffers, and 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 has been used to isolate mRNA from as little material as a single cell. Dynabeads® mRNA DIRECT™ Kit can be used for samples up to 200–400 mg tissue/20 x 10^6 cells. The protocol can be scaled up or down to suit your specific needs.
Yes, Dynabeads® Oligo(dT)25 magnetic beads can be reused. Please see the conditions we suggest below:
For reuse of the 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.
For reuse of a different sample:
To avoid carryover of mRNA between samples, the beads should be washed three times in 200 μL Reconditioning Solution by standard magnetic separation. Incubate at 65°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 2–8°C. Do not mix regenerated beads with the original stock suspension.
Dynabeads® magnetic beads are compatible with TaqMan® real-time PCR chemistry and non-capillary real-time PCR 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.
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., Nucleic Acids Research 25 (17), 3465–3470 (1997).
It is also possible to isolate RNA by specific capture using Dynabeads® Streptavidin magnetic beads with a biotinylated probe.
Their capacity is 2 μg per milligram of beads.
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.
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 in house with oligo(dT)25 on the beads as a primer. The cDNA synthesis was performed according to the manufacturer's instructions. When using a thermostable reverse transcriptase and the oligo(dT)25 primer for first-strand cDNA synthesis, an initial step of incubation at 50°C for 5 minutes 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 detach from 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.
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).
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.
No, Dynabeads Oligo(dT)25 magnetic beads allows direct use in PCR reactions. But it is not recommended to use more than 100 μg Dynabeads® Oligo (dT)25 magnetic beads per 50 μL total PCR reaction volume.
Dynabeads® DNA DIRECT™ Kits
The yield will depend on the number of nucleated cells present in the sample. One unit (200 µL) of Dynabeads® DNA DIRECT™ Universal Kit will isolate at least 200 ng of high quality, PCR-ready genomic DNA per sample, sufficient for 10 PCR reactions. The size of the sample used should be adjusted to match the Dynabeads® magnetic beads capacity. A typical starting sample is 10,000 cultured cells or 10 µL blood. The protocol can be adjusted to support 30 µL blood samples, sufficient for 30–50 downstream PCR amplifications (600 ng to 1 µg DNA).
The isolated DNA is maintained at chromosome length during the lysis and washing steps. During these steps, it is important to take care not to break up the DNA/Dynabeads® complex. The amount of shear force applied during resuspension determines the final average fragment length. Normally, the average fragment length exceeds 20 kb. The PCR results show excellent reproducibility in combination with a high level of sensitivity.
Dynabeads® DNA DIRECT™ magnetic beads are not reusable. It will never be possible to remove all traces of DNA from the magnetic beads. As with used tubes and pipette tips, traces of DNA on the beads will contaminate downstream PCR on any future samples.
Use a low ionic strength buffer that is slightly basic and heat (55 to 90°C).
That depends. If you want to isolate intact nucleosomes (histones + DNA complex), that is not possible with Dynabeads® SILANE beads. But you can isolate DNA from nucleosomes if they are not cross-linked. For chromatin immunoprecipitation, you could consider the MAGnify™ Chromatin Immunoprecipitation System.
The Dynabeads® SILANE Viral NA Kit will bind genomic DNA as well as viral nucleic acids, but the buffers have been optimized for stronger binding of shorter fragments and very small amounts of viral DNA/RNA. This buffer is not advised for genomic DNA binding, because the interaction with the bead surface will be stronger, which will require more work to elute genomic DNA than viral nucleic acids.
For Research Use Only. Not for use in diagnostic procedures.