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1. Freeze and grind up to 500 mg fungal sample under liquid nitrogen. Grind tissue completely to obtain a fine homogenous powder.
2. Immediately add 2.5 ml Buffer RB/2-mercaptoethanol. Add 10 ul 2-mercaptoethanol per 1ml of Buffer RB and then add 2.5ml of this mixture to the sample. Samples should not be allowed to thaw before Buffer RB/2-mercaptoethanol is added. Vortex vigorously to make sure that all clumps are dispersed. RNA cannot be effectively extracted from clumped tissue.
3. Centrifuge the lysate at 4,000 x g for 10 min at room temperature. Transfer the supernatant directly into a Homogenization Midi Column placed in collection tube. Centrifuge at 4,000 x g for 5 min at room temperature.
4. Carefully transfer the supernatant of the flow-through fraction to a new 15 ml centrifuge tube, making sure not to disturb the pellet or transfer any debris. Add equal volume of 70% ethanol (room temperature) and mix by vortexing at maxi speed for 20 seconds. If precipitation can be seen at this point, break the precipitation by passing through a needle using a syringe.
TIP: In most cases, 2.0 ml supernatant can easily be removed. This will require 2.0 ml of 70% ethanol. 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 ethanol.
5. Apply the entire mixture, including any precipitates that may form to a HiBind® RNA Midi column assembled in a 15.0 ml collection tube (supplied). Close the cap gently. Centrifuge at ?4,000 x g for 5 minutes at room temperature. Discard the flow-through liquid and place the column back into the collecting tube.
Optional on-membrane DNase I digestion: This is the starting point to perform DNase I digestion.
6. Add 3.5 ml RNA Wash Buffer I, close the tube gently. Centrifuge at?4,000 x g for 2 minutes. Discard both flow-through liquid and collection tube.
7. Place column in a clean 15 ml collection tube (Not supplied), and add 3.5 ml RNA Wash Buffer II diluted with absolute ethanol. Close the column gently, Centrifuge at 4,000 x g for 2 minutes at room temperature and discard flow-through. Re-use the collection tube in step 8.
Note: RNA Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions
8. Wash column with a second 3.5 ml of RNA Wash Buffer II by repeating step 7. Centrifuge as above and discard flow-through. Then with the collection tube empty, centrifuge the Midi column for 10 min at >4000 x g to completely dry the HiBind™ matrix.
9. Elution of RNA. Transfer the column to a new RNase-free 15 ml centrifuge tube (not supplied with kit) and elute the RNA with 0.3-0.5 ml of DEPC water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge at 4,000 x g for 5 minutes. A second elution into the same tube may be necessary if the expected yield of RNA >0.5mg.
Note: RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. No RNA extraction procedure can completely remove genomic DNA. For sensitive work (such as RT-PCR or differential display) we suggest that you treat the eluted RNA with RNase-free DNase. Also for RT-PCR, use intron-spanning primers that allow easy identification of DNAcontamination.
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