Biotinylated oligodeoxythymidine-streptavidin magnetic bead purification band Poly(A)+RNA
Biotinylated oligodeoxythymidine-streptavidin magnetic bead purification band Poly(A)+RNA
The use of magnetic microspheres instead of transferring fiber cords as a medium is more commonly used and has been commercialized.
Operation method
Biotinylated oligodeoxythymidine-streptavidin magnetic bead purification band Poly(A)+RNA
Materials and Instruments
GTC extraction buffer Dilution buffer β-mercaptoethanol No RNase Water 20XSSC 0.5XSSC 1XPBS. Move I MATERIALS AND EQUIPMENT Caveat 1) β-mercaptoethanol is not toxic and should be prepared in a fume hood with appropriate protective equipment.2) Magnetic beads may remain in the eluate, centrifuge at 12000g for lmin, transfer supernatant to a new tube. However, residual beads do not inhibit most of the conventional enzyme reactions used in RNA use and they often help to localize the precipitate after precipitation. For more product details, please visit Aladdin Scientific website.
Affinity-Specific Magnetic Beads (SA-PMP) Biotin-labeled oligo(dT) probe Magnetic rack 75°C water bath 50 ml sterile Corex centrifuge tubes 15 ml screw-cap conical centrifuge tubes TissuemizerDM or other comparable homogenizer BeckmanJ2-21 centrifuge or other comparable equipment
1) Affinity-Specific Magnetic Beads (SA-PMP)
2) GTC Extraction Buffer:4moL/I, Guanidine Isothiocyanate, 25 mmol/L Sodium Citrate, pH:7.1
3) Biotin-labeled oligo(dT) probe (50pmol/ul)
4) Dilution Buffer: 6XSSC, lmmol/L Tris-HCl, pH 7.4 10 mmoL/L EDTA, 0.25% SDS
5) β-mercaptoethanol (48.7%)
6) RNase-free water
7) 20XSSC: 67.7 g NaCl and 44.lg sodium citrate dissolved in 400 ml of deionized water, pH adjusted to 7.2 with NaOH, water added to a volume of 500 ml, treated with 0.1% DEPC overnight, autoclaved
8 ) 0.5XSSC: 20XSSC diluted in RNase-free water
9) 1XPBS
10) Magnetic rack
11) 75°C water bath
12) 50 ml sterile Corex centrifuge tube
13) 15 ml Screw-cap conical centrifuge tube
14) Tissuemizer DM or other homogenizers of the same type
15) Beckman J2-21 centrifuge or other similar equipment
II. Procedure
The purification of mRNA from tissues weighing less than 0.125 is an example, while the purification of mRNA from a large number of tissues and cells can be done by referring to the relevant product manuals.
1) Remove the GTC extraction solution, the biotin-labeled oligo(dT) probe, the RNA-enzyme-free water, and the 0.5 X SSC from the refrigerator, equilibrate to room temperature, and preheat the dilution solution at 70°C.
2) In a 50 ml sterile screw-cap conical centrifuge tube, start with a 50 ml sterile screw-cap tube. In a 50 ml sterile screw-cap centrifuge tube, first add lml of extraction solution (extraction/BME buffer) and then add 41ul of β-mercaptoethanol (48.7%) to ensure that the final concentration of β-mercaptoethanol is 2%. Weigh the tube with buffer and record.
3) Obtain the desired amount of tissue as quickly as possible and place it into the tube with extraction/BME buffer and homogenize the tissue at a high rate using a small homogenizer until there are no visible tissue clumps. If a homogenizer is not available, the tissue can be quickly minced, frozen in liquid nitrogen, and mashed and ground in a mortar filled with liquid nitrogen. Transfer the liquid nitrogen and tissue to a sterile 50 ml screw-cap conical tube and add the extraction/BME buffer as soon as the liquid nitrogen has evaporated, so that it is completely mixed.
4) Weigh the bone containing the tissue and the extraction/BME buffer, subtracting the weight of the tube from step 2) and calculating the size of the block.
5) Referring to Fig. 2.1, determine the amount of biotin-labeled oligo( dT) probe and the amount of tissue to be used for the biotin-labelled oligo( dT) probe, taking into consideration the size of the tissue block calculated in step 4). dT) probe and the amount of affinity protein-magnetic beads (SA-PMP) as calculated in step 4).
6) Add 2 ml of preheated dilution buffer to a sterile tube and add 41ul of β-mercaptoethanol (48.7%) to a final concentration of 1%. Add this solution to the homogenate. Mix completely. Add the probe identified in step 5, shake to mix thoroughly, and incubate at 70°C for 5 min.
7) Transfer the homogenate to a clean, sterile 15 ml Corex tube and centrifuge at 12,000 g for 10mim at room temperature.
8) While centrifuging, gently shake to keep the SA-PMP fully suspended and transfer the desired amount of beads to a sterile 50 ml screw-cap pyramid tube, which is then placed in a magnetic rack. Place the tube on the magnetic rack horizontally until the beads are all clustered on one side of the tube. Tilt the tube so that the solution does not flow over the surface of the beads. Carefully pour off the storage solution
9) Suspend SA-PMP with an equal volume of 0.5XSSC, capture with the magnetic rack and pour off the SSC solution as in step 8. Repeat the wash twice and resuspend SA-PMP in an initial volume of 0.5XSSC.
10) After centrifugation of the homogenate is complete, carefully remove the supernatant to avoid aspiration of the precipitate. Add the clarified homogenate to the tube containing the washed SAPMP and mix by turning.
11) Incubate the homogenate and SA-PMP at room temperature with a magnetic rack to capture the SAPMP until the homogenate is clear. Carefully decant the supernatant as described above. Collect the supernatant in a sterile tube and keep on ice until you are sure that the mRNA is satisfactorily bound and eluted.
12) Suspend the beads in lml of 0.5XSSC and transfer to a 2 ml tube. Capture the beads with a magnetic rack, carefully decant the SSC and repeat the wash twice. After the last wash, remove as much SSC as possible without stirring the SA-PMP.
13) Elute the mRNA with 0.5 mL of nuclease-free water, and resuspend the SA~PMP by gently shaking the tube.
14) Capture the SA-PMP with the magnetic rack, and transfer the eluted RNA to a small, rinseless centrifuge tube. Resuspend magnetic beads in sterile water and keep on ice until mRNA yield and purity are determined.
15) Add 0.1x volume of NaAc (for cDNA cloning) or KAc (for in vitro translation) and 1x volume of isopropanol to the eluate and let stand overnight.
16) Centrifuge at 12,000 g for 10 min, resuspend RNA precipitate with 1 ml of 70% ethanol, and then re-centrifuge
17) Vacuum dry precipitate for 15 min. Dry the precipitate for 15 min and dissolve the precipitate in RNase-free deionized water.