RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation

3 RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation 

Prepare a linear gradient from Soln. B and Soln. C in a centrifuge tube (e.g., 13 ml Ultra-Clear for Beckman SW 41 rotor 6 ml of Soln. B and Soln. C each) at the day before run. If a gradient mixer is used, e.g., according to Fig. 2.2, place tbe centrifuge tube either in a slanting position, start with Soln. C, and let flow along the tube 

Cover the sucrose gradientwithO.lOmlRNA-containingsample and precisely tare the tubes which will be opposite in the rotor. 

Spin the samples with 200 000 x g at 4 °C for 15 -17 h. After the run, displace the gradient using a more dense solution, e.g., 40% sucrose in Soln. A, colored by a droplet Amido Black 10 B solution. The principle of a displacement apparatus is shown in Fig. 5.4. The RNA content of the fractions is measured either by reading the UV absorption at 260 nm or, if labeled material was used, by counting the radioactivity. To monitor the sucrose gradient, estimate the refractive index of the obtained fractions (concentration, density and refractive index of sucrose solutions are given in Table 8.17). 

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