1. Prepare a 6% acrylamide gel in TBE, with 7 M urea. Two mini-protean II gels are sufficient to purify satellite from about 80 µg of viral RNA. Use a prep style comb for most efficient results.
  1. Viral RNA should be resuspended in 0.1 mM EDTA and stored at -20°C. Mix the viral RNA at about 1 mg/ml with an equal volume of formamide dye (99% deionized Formamide; 0.1 mM EDTA; 0.025% brome phenol blue and xylene cyanol).

NOTE: you do not need to heat the samples before running, the urea and formamide will partially denature the RNA without heating, and it will run as a nice sharp band.

  1. Load the gel with 40 µg viral RNA. Run at 250 volts until the brome phenol blue dye runs completely off the bottom and the xylene cyanol is a few centimeters from the bottom (about 30 to 45 minutes).
  1. Disassemble the gel apparatus. Leave the gel on one glass plate and place on a tray that can catch run off (we usually use an empty pipette tip box). Stain the gel by flooding with about 5 ml of Toluidine blue dye for 1 minute. Destain by flooding with milli-Q water until the bands appear. It is easiest to use a light box to visualize the bands while cutting them out, but minimize light exposure to prevent damage. Excise the satellite band with a fresh scapel blade, it will be about halfway down the gel. The viral RNA bands will be unseparated, and near the top of the gel. RNA 4 may separate somewhat from the others, but the satellite will be the lowest band in the gel.
  1. Place the excised bands in a baked 15 ml COREX tube, and add 2.5 ml of elution buffer per gel (0.5 M NH4OAc; 0.1% SDS; 1 mM EDTA). Place in a dark place overnight.
  1. Remove the eluate being careful not to take any of the gel. The eluate can be passed through a plug of siliconized glass wool to remove excess acrylamide. Extract the eluate with 1:1 phenol:chloroform. Add 1/10th volume 3M NaOAc and ethanol precipitate. The precipitate should be spun in a swinging bucket rotor if at all possible to minimize loss of the rather small pellet. Resuspend thoroughly in 0.5 ml of NAE (0.3 M NaOAc pH 6; 0.1 mM EDTA), and re-precipitate in an eppendorf tube. The pellets may look bigger than expected, this is due to some residual acrylamide.
  1. Yield should be about 10% of viral RNA.

NOTE: This RNA should be treated as an infectious pathogen!

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