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œœœœœ@5' End-labeling Alkaline Hydrolyzed rRNA@œœœœœ



The best way to obtain high specific activity labeled rRNAs is to hydrolyze the RNA by alkaline treatment at high temperature, followed by 5' end-labeling with PNK & gamma-32P-ATP. This way, you get many 32P's incorporated into each rRNA molecule. This is also useful in heterologous probings, because the similarities of some of the RNA fragments will be much higher than the overall similarity for the heterologous rRNAs.
After alkaline hydrolysis, phosphatase treatment is not needed because the fragments contain 5' hydroxyl's and 3' phosphates.
REMEMBER TO USE RNase-FREE TECHNIQUE, ESPECIALLY AFTER THE HYDROLYSIS STEP. USE ONLY DEPC-TREATED H2O.

  1. Materials

    1. 100mM Tris, pH 9.5

    2. Denaturation buffer
      Final Conc.
      10ul 1M Tris, pH8 10mM
      1ul 100mM EDTA 0.1mM
      7.5ul 0.19mg/ml spermidine-3HCl 100mM
      1ml

    3. 10X PNK buffer
      0.5M Tris, pH9.5
      0.1M MgCl2
      50mM DTT
      50% glycerol

  2. Methods

    1. Dissolve 5-10ug of ribosomal RNA in 25ul ddH2O. Add 25ul 100mM Tris, pH9.5 and incubate at 95C for 3min.
    2. Add 70ul denaturation sol'n, & heat for 3 min. at 50C. Chill on ice.
    3. Working on ice, mix in:
      148ul ddH2O
      0.5ul (75uCi) gamma-32P-ATP
      30ul 10X PNK buffer
      10units (1-2ul) polynucleotide kinase

    4. Incubate 37C 30min., then add 33ul 3M Na acetate and 660ul EtOH & incubate 5min. in a dry ice:EtOH bath. Centrifuge 5min., drain, and dry the RNA pellet.
    5. Dissolve in 100ul ddH2O. Count 1ul to to determine incorporation. Store at -20C.



Elizabeth S. Haas, personal communication
Charles J. Daniels, personal communication

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