By Dr. Felipe Sierra (auth.)
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B. Clean nuclei obtained after a single ultracentrifugation through a sucrose cushion. C. Nuclei are contaminated by red blood cells and other debris after the first centrifugation, due to an excessive amount of tissue being processed at one time. D. Nuclei are contaminated by cytoplasmic tags due to insufficient homogenization. III Experimental 39 homogenization buffer containing an additional 10% of glycerol. Usually, this step is carried out in about 50 ml (two SW27 tubes). The nuclei are resuspended by bubbling and scraping the side of the tube with a plastic pipet (avoid glass pipets, as nuclei stick much more to them), then, carefully aspirate up and down several times, making sure not to produce foaming.
There are many rapid methods for preparing reasonably clean DNA; here, I give the protocol I use routinely to prepare DNA to be used as template for in vitro transcription reactions (in fact, I use the same DNA preparations for all other purposes as well). III Experimental 53 Materials and Buffers > 37°C shaker incubator > 250-ml flasks with cotton plugs > High speed centrifuge > Swing out rotor (50-ml tube capacity) > 37°C water bath > Plastic 10-ml pipets > Siliconized glass wool > Mini gel and power supply Chemicals: > L-broth or other liquid media for bacterial growth > 10 mM Tris .
Add 100 III of 2 x Stop Buffer: 2 x: 20 mM Tris . 3MNaCI Heat at 65°C for 5 minutes. Extract twice with phenol/chloroform. Precipitate with 500 III of ethanol (no more salt required). - Wash RNA pellet in 70% ethanol and dissolve in H20 without drying the pellet. This RNA is ready for further analysis. Addendum 2: Preparation of Polyacrylamide/Urea Gels Since these gels are routinely used for many analyses in molecular biology (sequencing and other methods), I will not give a detailed description on how to prepare the gels.
A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)