Denaturating RNA Gradient Centrifugation

Fill 1/5 of the effective volume of a centrifuge tube with Soln. B, cover it with the same volume of Soln. C, followed by Soln. D and Soln. E. Store the centrifuge tubes vibrationless at RT overnight. A linear gradient is formed by diffusion during this time. 

Mix 60 (xlofRNA solution with 275 jilDMSO, followed by 165 jil DMF. Cover each gradient with 200 jil of this RNA solution. 

Spin the samples with 200 000 x g at 25 °C for 15 -17 h. RNA monitoring by UV reading is not possible, because DMSO absorbs below 275 run, i.e., within the range of the nucleic acid absorption. 

Rickwood D (ed.) (1992) Preparative centrifugation a practical approach. Oxford University Press, London 

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