互联网2013-11-13
1. Grind plant sample. Collect frozen ground plant tissue (up to 500 mg) in a microfuge tube and immediately add 3.5ml Buffer RPL/2-mercaptoethanol. We recommend starting with 250 mg tissue at first. If results obtained are satisfactory increase amount of starting material. Add 20 ul 2-mercaptoethanol per 1ml of Buffer RPL. Samples should not be allowed to thaw before Buffer RPL/2-mercaptoethanol is added. Vortex vigorously to make sure that all clumps are dispersed. RNA cannot be effectively extracted from clumped tissue.
Note: Add 20 ul 2-mercaptoethanol per 1 ml of Buffer RPL before use. This mixture can be made and stored at room temperature for 1 week.
2. Add 700 ul Buffer SP and vortex thoroughly to mix. Centrifuge at 4,000 x g for 20 min at room temperature.
3. Carefully aspirate cleared lysate to a RNase-free 50 ml centrifuge tube making sure not to disturb the pellet or transfer any debris. Add one volume isopropanol and vortex to precipitate RNA. This step removes much of the polysaccharide content and improves spincolumn performance by increasing RNA binding capacity (and therefore yield) in the steps that follow. No incubation is required after addition of isopropanol.
TIP: In most cases 3 ml supernatant can easily be removed. This will require 3 ml isopropanol. Note that depending on the sample, the volume of supernatant may vary. After transferring to a fresh tube, measure the volume and add the correct amount of isopropanol.
4. Immediately centrifuge at 4,000 x g for 20 min at room temperature to pellet RNA. A longer centrifugation does not improve yields.
5. Carefully aspirate or decant the supernatant and discard making sure not to dislodge the RNA pellet. Invert the microfuge tube on a paper towel for 5 min to allow residual liquid to drain. Drying the pellet is not necessary.
6. Add 500 ul of RB buffer pre-heated to 65℃ and vortex to resuspend the pellet. A brief incubation at 65℃ may be necessary to effectively dissolve the RNA.
7. Add 1.25ml Buffer RB/2-mercaptoethanol followed by 900 ul absolute ethanol. Vortex thoroughly to mix. This will adjust binding conditions prior to loading the HiBind® RNA column.
8. Apply the entire sample, including any precipitates that may form to an HiBind® RNA Midi column assembled in a clean 15 ml collecting tube (supplied). Centrifuge at 4,000 x g for 5 minutes at room temperature. Discard the flow-through liquid and place the column back into the collection tube.
Note: This is the starting point to perform DNase I digestion.
9. Add 3 ml RNA Wash Buffer I and centrifuge at 4,000 x g for 5 minutes. Discard both flow-through liquid and collecting tube.
10. Place column in a clean 15ml collection tube (Not supplied), and add 3.5 ml RNA Wash Buffer II diluted with ethanol. Centrifuge at 4,000 x g for 5 minutes at room temperature and discard flow-through. Reuse the collection tube in step 11.
11. Wash column with a second 3 ml of RNA Wash Buffer II as in step 10. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the Maxi column at 4000 x g for 10 minutes to completely dry the HiBind™ matrix.
12. Elution of RNA. Transfer the column to a RNase-free 15 ml centrifuge tube (not supplied with kit) and elute the RNA with 0.5 ml of DEPC water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge at 4000 x g for 5 minutes at room temperature. A second elution into the same tube may be necessary if the expected yield of RNA >0.5 mg.
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