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Because RNase P in eubacteria is essentially an RNA molecule (with a non-essential protein helper), RNase P RNA can be purified using standard methods for the purifiction of RNA molecules, with the help of an enzyme assay to test for the presence of the P RNA. The purification scheme described here has been successfully used to purify RNase P RNAs from several organisms of sufficient purity for use as probes to identify the genes for these RNAs, or for biochemical examination of the RNA catalyst itself.
In the process of the purification, you also get as byproduct 23+16S rRNA, which can, & should, be used at 25ug/ml in hybridization experiments when using purified P-RNA as probe.
Remember to be careful of RNase - use disposable plastics whenever possible & absolute EtOH. The column should be prepared in advance, & is poured, equilibrated, run & stored at room temperature in STE-SDS (SDS inhibits any RNases & microbial growth).

  1. Materials

    1. STE-SDS
      100mM NaCl
      10mM Tris, pH7.5
      1mM EDTA
      0.1% SDS

    2. Elution buffer
      0.5M NH4OAc
      10mM Mg(OAc)2
      1mM EDTA
      0.1% SDS

    3. 3M NaOAc

    4. Gel sample buffer
      50% urea
      1X TBE
      0.025% bromophenol blue
      0.025% xylene cyanol FF

    5. all the reagents needed for urea-PAGE
      all the reagents needed for RNase P assays (including substrate)

  2. Methods

    1. Dissolve 10mg of total cellular RNA in 2ml STE-SDS, and add 100ul glycerol. For best resolution, do NOT preheat the sample!
    2. Using a plastic pasteur pipette, carefully load the sample onto the top of a 120ml sepharose 4B column (1.5 X 68cm). Since the sample will be quite dense, there is no need to remove the buffer from above the column - just load "inject"it under this buffer just like loading a gel. The column should be equilibrated in STE-SDS at room temperature & rinsed well before use.
    3. Run the column overnight with STE-SDS at room temperature, collecting 3ml fractions.
    4. Measure the A260 of each fraction (use a 2mm thick cuvette to eliminate the need to dilute fractions), and run alternate fractions on a 4% denaturing acrylamide gel. Stain the gel with ethidium bromide & photograph.
    5. Assay fractions between the void volume peak (23S+16S rRNA) and the included peak (5S rRNA, tRNA, trash) for RNase P activity in RNA-alone conditions (use 3-5ul of each fraction). Pool the most active fractions and precipitate by the addition of 0.1volume 3M NaOAc and 2.5 volumes EtOH at -70C >2hr. Collect the RNA by centrifugation (10KRPM, 30minutes, 4C, HB4 or SS34 rotor), and drain the pellet dry. Save the void volume peak fractions (EtOH precipitate) for use in hybridization experiments as rRNA "blocker".
    6. Dissolve the RNA pellet in 500ul urea-sequencing sample dye and incubate for 3 minutes at 65C & quick-cool on ice. Load the sample onto a 3mm thick 4% denaturing acrylamide prep gel (10cm wide "comb") & electrophorese until the xylene cyanol dye almost reaches the bottom of the gel.
    7. Examine the gel by UV shadowing with a PEI plate. Using a NEW single-edged razorblade, excise each visible band from the gel & elute each overnight at 4C in 2ml elution buffer with constant rotation.
    8. Collect the eluted RNA from each sample & precipitate the RNA by adding 6ml EtOH & incubating at -70C for 2hr. Pellet the RNA at 10KRPM, 4C, 30 min., HB4 rotor, drain the pellets, rinse twice with -20C 70% EtOH, and dissolve in 250ul STE-SDS. AAssay 1ul aliquots of each fraction for RNase P activity & store at -70C.


JWBrown, unpublished data

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