New PDF release: A Laboratory Guide to In Vitro Transcription

By Dr. Felipe Sierra (auth.)

ISBN-10: 3034863837

ISBN-13: 9783034863834

ISBN-10: 3764323574

ISBN-13: 9783764323578

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Additional resources for A Laboratory Guide to In Vitro Transcription

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This way it is possible to diminish losses, and there are usually no problems of reproducibility. It should be mentioned that not all tissues have the same ratio of DNA/nuclear proteins (for example, nuclei from spleen and livers from old animals have less protein) and not all tissues have the same number of nuclei per gram of tissue. Thus, the yield of nuclear proteins from tissues such as kidney or salivary glands is about 1/5 of that from young liver, even though the DNA/protein ratios are similar.

Homogenization is done in a modified food processor designed in U. Schibler's laboratory (Figures 4 and 33). This allows for faster and easier manipulations at this critical step. Unfortunately, it has not been possible to avoid the use of the ultracentrifuge, and the relatively low capacity ofthe tubes makes this step the most time consuming. Nevertheless, for preparative purposes, spinning just once, and for only 40 minutes seems to be adequate, since further chromatographic purifications will be introduced later.

Cytoplasm Figure 5. Appearance of the Ultracentrifuge Tube After Spinning. The diagram shows the approximate appearance ofthe tubes, as well as the localization of several cellular and subcellular compO{lents. Lysed Nuclei .... Clean Nuclei III Experimental 37 Whole cells and unbroken pieces of tissue float. Nuclei with cytoplasmic tags and broken nuclei stay at the interface. Clean nuclei pellet. > While spinning, check a small aliquot of material under the microscope. Most cells must be broken, but most nuclei should be intact.

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A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)

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