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Oligo dT25 Magnetic Beads for mRNA purification (2 ml)

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Oligo dT25 Magnetic Beads mRNA purification

Oligo dT25 Magnetic Beads – 25 mg. Oligo d(T)25 Magnetic Beads are 100 nm Fe3O4 based for the small-scale isolation of mRNA from crude cell lysates and tissue. The isolation occurs through the complement of covalently coupled oligo d(T)25 to the poly(A) region present in most eukaryotic mRNA.

Oligo dT25 magnetic bead is covalently coupled to 100 nm Fe3O4 magnetic beads through a linkage that is stable and leak-resistant over a wide pH range, thereby permitting the small-scale isolation of mRNA from crude cell lysates and tissue. The isolation occurs through the hybridization of covalently coupled oligo d(T)25 to the poly(A) region present in most eukaryotic mRNAs.

Applications include direct mRNA isolation following lysis and second-round purification of previously isolated total RNA. The magnetic separation technology is scalable and permits elution of intact mRNA in small volumes eliminating the need for precipitation of the isolated mRNA. Beads can be reused up to three times and the researcher has the option of eluting the isolated mRNA or using the bound d(T)25 as a primer in a first-strand cDNA reaction.

• Allows for direct mRNA isolation following lysis and second-round purification of previously isolated total RNA
• The magnetic separation technology is scalable and permits elution of intact mRNA in small volumes, thereby eliminating the need for precipitation of the isolated mRNA
• Beads can be reused up to three times
• The isolated mRNA can either be eluted or the bound d(T)25 can be used as a primer in a first-strand cDNA reaction

  • Step 1
    Aliquot the appropriate volume of Oligo d(T)25 Magnetic Beads for the scale of isolation (Table 1). Add 200 µl of Lysis/Binding Buffer to beads, vortex briefly, and mix with agitation for 2 minutes. Beads should remain in the lysis/binding wash solution until removal immediately before adding the cell lysate.
    Step 2
    •    Adherent Cells
    1. Aspirate media from the cell culture plate and wash once with cold sterile 1X PBS (pH 7.4).
    2. Add the appropriate volume of Lysis/Binding Buffer (see Table 1) to cells and gently swirl by hand.•    Suspension Cells
    1. Pellet cells by centrifuging at 1,000 rpm for 5 minutes at 4°C.
    2. Aspirate media and wash once with cold sterile 1X PBS (pH 7.4).
    3. Pellet again, discard PBS and add the appropriate volume of Lysis/Binding Buffer (Table 1) to cells.
    4. Agitate to suspend cells in Lysis/Binding Buffer.
  • Step 3
    Incubate at RT for 5 minutes with gentle agitation. If the solution is viscous, pass the lysate several times through a 21-gauge needle attached to a 1 or 2-ml syringe. A noticeable decrease in viscosity should be observed.
    •Place the microcentrifuge tube containing the beads and lysis/binding wash into the magnetic rack and pull the magnetic beads to the side of the tube.
  • Step 4
    •    Remove Lysis/Binding wash and add the cell lysate to the equilibrated magnetic beads.
    •    Place cell lysate-and-bead suspension on the agitator and incubate at RT for 10 minutes.
    •    Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard the supernatant.Step 5
    •    Add the appropriate volume of Wash Buffer 1 (see Table 1) to the beads and mix with agitation for 1 minute.
    •    Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
    •    Repeat step 5, once.Step 6
    •    Add the appropriate volume of Wash Buffer 2 (see Table 1) to the beads and mix with agitation for 1 minute.
    •    Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
    •    Repeat step 6, once.Step 7
    •    Add the appropriate volume of Low Salt Buffer (Table 1) to the beads and mix with agitation for 1 minute.
    •    Place microcentrifuge tube in the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.Step 8
    •    Add the appropriate volume of Elution Buffer (Table 1) and vortex gently to suspend beads.
    •    Incubate at 50°C for 2 minutes with occasional agitation to elute poly(A)+ RNA. This step enables > 90% of the poly(A)+ RNA bound to the beads to be recovered.
    •    Place the microcentrifuge tube in the magnetic rack and pull the magnetic beads to the side of the tube. Transfer eluent to a clean, sterile RNase-free tube and store on ice for immediate quantitation or place at –80°C for long-term storage.
  • Table 1: Recommended volumes for different isolation scales (for cells).
NUMBER OF CELLS VOLUME OF OLIGO D(T)25 BEADS VOLUME OF LYSIS/BINDING BUFFER VOLUME OF WASH BUFFERS & LOW SALT BUFFER VOLUME OF ELUTION BUFFER EXPECTED YIELD (µg)
5 x 104 50 µl 250 µl 250 µl 50 µl ~0.5–2
1 x 105 100 µl 500 µl 500 µl 100 µl ~1–3
5 x 105 100 µl 500 µ 500 µl 100 µl ~2–4

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