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œœœœœ@Preparing 5' end-labeled alkaline hydrolyzed RNA@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. 10X Kinase buffer
      0.5M Tris,pH9.5
      0.1M MgCl2
      50mM DTT
      50% glycerol

    2. TE-SDS
      10mM Tris, pH8
      1mM EDTA
      0.1% SDS

  2. Methods

    1. Dilute 0.1-5 ug of RNA to 120ul in ddH2O, then divide into 4 30ul aliquots.
    2. Add 30ul 100mM NaHCO3, pH9, to each sample and start the incubation at 95C. Remove one tube at 5 min. intervals (i.e. at 5, 10 15 and 20 min.) & transfer to ice.
    3. Add 6ul 3M NaOAc and 180ul EtOH to each tube and pool the samples. Incubate in a dry ice:EtOH bath for 10min., centrifuge 10min., drain and dry the samples in a rotovac.
    4. Redissolve the dry sample in:
      5ul 10X PNK buffer
      1ul (100uCi) gamma 32P-ATP
      43ul ddH2O
      1ul Polynucleotide kinase

    5. Incubate at 37C for 30min., then add another 1ul of kinase & incubate 37C for 30min.
    6. Add 10ul 100mM EDTA and 5ul of column dye. Separate the sample on a G50 column (in TE-SDS), collecting 20 15drop fractions.
    7. Count 1ul of each fraction, & pool the highest fractions from the first peak. Store at -20C.


Bryan James, personal communication

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