Product Literature

Brochure: RNA isolation and purification (non-North America)

Product Literature

Brochure: RNA isolation and purification (North America)

Product FAQ

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

Answer

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.

Answer Id: E4780

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Product FAQ

Could you suggest references for cDNA libraries and RT-PCR using Dynabeads magnetic beads?

Answer

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

Answer Id: E6181

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Product FAQ

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

Answer

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.

Answer Id: E7945

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Product FAQ

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

Answer

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.

Answer Id: E4759

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Product Literature

Sera-Mag Magnetic Particles Oligo(dT)-Coated

Product FAQ

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

Answer

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.

Answer Id: E7946

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Product FAQ

Can Dynabeads Oligo(dT)25 magnetic beads be reused?

Answer

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.

Answer Id: E6066

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Product FAQ

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

Answer

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.

Answer Id: E4776

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Product FAQ

Can genomic DNA and mRNA be isolated from the same sample with Dynabeads magnetic beads?

Answer

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).

Answer Id: E6073

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Product FAQ

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

Answer

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.

Answer Id: E7937

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Product FAQ

What is the capacity of Dynabeads Oligo(dT)25 magnetic beads?

Answer

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.

Answer Id: E6069

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Product FAQ

Is it possible to generate a full-length cDNA from mRNA attached to Dynabeads magnetic beads, and what reverse transcription kit do you recommend?

Answer

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.

Answer Id: E6078

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Product FAQ

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

Answer

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.

Answer Id: E4772

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