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RNA Cleaning (mini)

  1. Add 500 µl Buffer LY (add b-mercaptoethanol before use) to the reaction (up to 100 µl).

  2. Add 1/2 volume 100% ethanol into the mixture (for example: 250 µl 100% ethanol for 500 µl mixture) and pipet 5 times to mix the solution. Vortex briefly if any precipitations.

  3. Transfer the solution into the binding column and centrifuge at 14,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.

  4. Add 400 µl RNA Wash Buffer (add ethanol before use) to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

  5. Optional: Add 50 µl DNase I (5URNase-free) solution onto the middle of the column and incubate at room temperature for 15 min. Add 200 µl DNase Stop Buffer onto the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through. Add 300 µl RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

  6. Add another 400 µl RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 30 s. Discard the flow-through.

  7. Centrifuge the column with the lid open at 14,000 rpm for 1 min. Discard the flow-through.

  8. Centrifuge at 14,000 rpm for another 1 min. It is critical to remove residual ethanol for optimal elution.

  9. Place the column to a RNase-free 1.5 ml tube and add 50-100 µl DEPC-treated water to the column and centrifuge at 14,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution at -200C.

Note: It is highly recommended that RNA quality be determined before downstream applications. The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining. Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling or storage, RNA molecule less than 200 bases in length do not efficiently bind to the RNA column. An A260/A280 ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.

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