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RNA Miniprep Protocol - Plant

  1. Weigh 30-100 mg plant tissue in a 2 mL tube. Freeze the plant tissue in liquid nitrogen and grind using a rotor-stator.

  2. Transfer 500 µL Buffer LY to the tube containing the plant tissue immediately. Grind using a rotor-stator again.Determine the volume of Buffer LY to be used and add 10 µL of β-mercaptoethanol (β-ME) per 1 mL Buffer LY before use. Buffer LY contains (β-ME) can be stored at room temperature for up to 1 month. Use of too much mass of tissue per preparation will cause genomic DNA contamination.

  3. Centrifuge the lysate for 5 min at 13,000 rpm at room temperature and transfer the cleared lysate to a clean 1.5 ml tube. 

  4. Add 1/2 volume of the 100% ethanol to the lysate (for example: 250 μl 100% ethanol for 500 μl lysate). 

  5. Transfer the solution into a RNA column and centrifuge at 10,000 rpm for 1 min. Discard the flow-through liquid.

 Without DNase I treatment:

  1. Add 400 µl Buffer RB to the column and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.

  2. Add 600 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol is added to RNA Wash Buffer before use.

  3. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.

  4. Transfer the column to an RNase-free 1.5 mL tube. Add 35-50 µl DEPC-treated ddH2O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.

With DNase I treatment:

  1. Add 500 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use.

  2. Add 75 µl DNase I (5URNase-free) with Digestion Buffer onto the middle of the column and incubate at room temperature for 15 min. Add 400 µl Buffer RB to the column, incubate for 1-2 min, and centrifuge at 10,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.

  3. Add 600 µl RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use.

  4. Centrifuge the column with the lid open at 10,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.

  5. Transfer the column to an RNase-free 1.5 mL tube. Add 35-50 µl DEPC-treated ddH2O to the center of the column. Centrifuge at 10,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.

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