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Usually, gel electrophoresis is used to analyse RNA according to size; however, sometimes sucrose gradient ultracentrifugation is preferable or necessary: For example, for preparative isolation of 4s or rRNAs, for tracing poly(A)-containing RNA with 3H-poly(U), for correlating CPM to S values, etc. The method described here allows the separation of\E.coli\RNAs into distinct, well isolated 4s, 16s, and 23s bands.
Naturally, strict RNase-free technique must be used. Autoclave all glassware and solutions (except sucrose) before use, and use virgin plasticware (including the tubing for collecting fractions!). Pretreat the centrifuge tubes and gradient maker with 0.3% DEPC for 2 hr at room temperature. DO NOT AUTOCLAVE NITROCELLULOSE CENTRIFUGE TUBES!


  1. Materials

    1. 50% sucrose
      50 grams sucrose plus 50ml ddH2O. Use RNase-free sucrose only. Sol'n is 50% w/w

    2. 10X TEN (per 100ml)
      conc.
      1.2114 grams Tris, pH 7.8 0.1M
      0.5844 grams NaCl 0.1M
      0.3802 grams Na4EDTA 10mM

    3. 5% sucrose in TEN (50ml)
      conc.
      5ml 50% sucrose 5%
      5ml 10X TEN 1X
      40ml ddH2O 10mM

    4. 20% sucrose in TEN (50ml)
      conc.
      20ml 50% sucrose 20%
      5ml 10X TEN 1X
      25ml ddH2O 10mM

  2. Methods

    1. Sample preparation:
      Dilute 50-300ug (1.5-8 A260 units)of total RNA to 0.5ml with TEN. If necessary, first EtOH precipitate the RNA, then redissolve in 0.5ml TEN. It's a good idea to run parallel gradients with two different amounts of RNA.

    2. Gradient preparation:
      Prepare two 17ml 5-20% linear gradients in SW27.1 tubes using 5% and 20% TEN buffered sucrose and a gradient maker. Premade gradients should be loaded and run immediately (within hours). Gradients can also be made by carefully layering 4.25ml layers of 20, 15, 10, then 5% TEN buffered sucrose on top of each other in the centrifuge tube. The handmade gradients should then be carefully stored overnight at 4C (don't slam the refrigerator door!) to allow the gradient to smooth out by diffusion.

    3. Ultracentrifugation:
      Very carefully layer the samples on top of the gradients, and balance with TEN. Run the gradients with slow acceleration (setting of 1-2), brake off, 27KRPM, for 25hr at 4C. When the rotor coasts to a stop, the w2t should be 7.1-7.2 x10/11 rad2/sec.

    4. Fractionation:
      Connect the tube puncturer outlet to a UV spectrophotometer flow-cell and fraction collector (or fractions can be collected manually). The readings on the spect. and stripchart should be: wavelength = 260nm, 1cm path length, small aperture, calibrated absorbance of 1 (for dilute samples) to 3 (for concentrated samples), chart speed of 1cm/min.
      The gradients should be fractionated as soon as possible, one after the other. Fill the tubing and flow-cell with 20% sucrose, to prevent gradient-stirring bubbles, and clamp off the tubing. Puncture the gradient tube and unload the gradients at 1-2 sec/drop. Drain the enire gradient, but do not let air into the flow-cell. Refill the tubing and flow-cell with 20% sucrose, and go to the next gradient. If a peristaltic pump is used to unload the gradients, more accurate fractions can be collected, but the flow-cell cannot be used.
      Fractions can then be analysed by scintillation spectrophotometry, absorbance (at some wavelength other than 260nm), gel electrophoresis, etc, and correlated to the A260 profile profile.
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