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RNA Miniprep Protocol - Bacteria

For Both Gram-positive (B. subtilis) Or Gram-negative (E. coli) Bacteria

  1. Grow an overnight bacterial culture in the appropriate media at an appropriate temperature. In the following day, dilute the culture 1:50 with media and grow until the OD600 reads at 0.6-1.0. This should only take a few hours. If the growth is too slow, reduce the dilution factor. Do not use the overnight culture for RNA isolation!

  2. Harvest no more than 3 ml culture ( < 5x108) by centrifugation at 3,000 rpm for 10 min.

  3. Carefully remove the supernatant as much as possible.

  4. Resuspend the pellet in 100 µl freshly prepared TE Buffer containing lysozyme. (Use 3 mg lysozyme per 1 ml TE Buffer for Gram-positive bacteria and 0.4 mg/ml lysozyme for Gram-negative bacteria). Mix by tapping gently. TE buffer: 10 mM Tris-HCl, pH 7.5; 1 mM EDTA.

  5. Incubate the resuspended pellet at room temperature for 5-10 min for Gram-positive bacteria, or 3-5 min for Gram-negative bacteria.

  6. Add 400 µl Buffer LY (add b-mercaptoethanol before use). Mix gently. Add 250 µl absolute ethanol (96-100%) to the lysate.

  7. 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.

Without DNase I treatment:

  1. Add 400 µl Buffer RB to the column and centrifuge at 11,000 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 11,000 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 11,000 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 50 µl DEPC-treated ddH2O to the center of the column. Centrifuge at 11,000 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 11,000 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 11,000 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 11,000 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 11,000 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 50 µl DEPC-treated ddH2O to the center of the column. Centrifuge at 11,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20°C.

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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