Download e-book for iPad: A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)

By Dr. Felipe Sierra (auth.)

ISBN-10: 3034863837

ISBN-13: 9783034863834

ISBN-10: 3764323574

ISBN-13: 9783764323578

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Extra resources for A Laboratory Guide to In Vitro Transcription

Example text

Whole Celis & Membranes .... 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.

1 --=----------'-L-I~----J C2 AT B pBR Hinf! 1 2 J -• - Figure 8. Transcripts Synthesized In Vitro from the G-free Cassette under the Control of Two Different Promoters. A. Diagram of the G-free cassette vector (C2AT) and the two promoter-containing constructs. The 5'-flanking region of each gene is shown either stippled (Adeno Major Late) or hatched (mouse albumin), and the sequences downstream of the cap site are represented in black. B. In vitro transcription products. The three templates just described were assayed in a standard in vitro transcription reaction using liver nuclear extracts, and the products were displayed on a 4% polyacrylamide/urea gel.

6 350 JlI 87% glycerol 85 JlI :5 M KCI 60 JlI 1 M MgCl2 250 JlI 25 mM CTP 250 JlI 25 mM ATP 35 JlI 10 mM UTP 200 Jll10 mM O-Methyl GTP 670 Jll H20 This solution should be stored frozen at -20°C, and it can be thawed and refrozen many times. 5 M This solution should be stored at room temperature. 60 III Experimental Step-by-Step Procedure In Vitro Transcription (with G-free Cassette) > Unless specified, all manipulations are carried out on ice. > The total volume of the reaction is 20 Ill. 8-1Ilg per reaction).

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


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