Download Chromosome Microdissection and Cloning. A Practical Guide by Nabil Hagag PDF

By Nabil Hagag

Chromosome Microdissection and Cloning: a realistic consultant is a simple advisor to chromosome microdissection and cloning. It provides an summary of the systems and in short studies a number of parts of study during which those thoughts are utilized. subject matters variety from instruction of chromosomes for microdissection to molecular cloning of microdissected chromosomal DNA. tools of chromosome microdissection, together with video microscope approach and oil chamber technique, are described.

Comprised of 5 chapters, this booklet starts off with an summary of the constitution and association of chromosomes, by way of an outline of equipment for getting ready and retaining chromosomal DNA in a fashion that's precious for cloning and direct research. Microdissection of metaphase chromosomes and isolation of fragments will be entire in a single of the 3 methods defined within the subsequent bankruptcy: by means of microdissection utilizing an upright microscope and glass capillaries in an oil chamber; through laser microbeam; and with using an inverted microscope outfitted with a video digital camera and excessive magnification-high answer lenses. A step by step consultant to those innovations and options for universal difficulties are given following every one process. Protocols for cloning and determining genetic sequences from outlined chromosome areas, relatively utilizing the polymerase chain response, also are mentioned. the ultimate bankruptcy specializes in purposes of chromosome microdissection, equivalent to cloning of disease-specific genes and producing "sequence tagged websites" for use in huge DNA sequencing projects.

This monograph may be fairly valuable to investigators developing microdissection platforms de novo.

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Extra info for Chromosome Microdissection and Cloning. A Practical Guide

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Cell Res. 167, 311-325. 14. Edström, J. , and Röhme, D. ) (1987). Microcloning of mammalian metaphase chromosomes. In "Methods in Enzymology," Vol. 151, pp. 503-516. Academic Press, New York. 15. Korthof, G. (1986). An improved fixation method for chromosome preparation of Chinese hamster-human hybrid and mouse cell lines. Cytogenet. Cell Genet. 41, 181-184. 16. Pardue, M. , and Gall, G. (1975). Nucleic acid hybridization to the DNA of cytological preparations. In "Methods in Cell Biology" (D. V.

11 Micropipette grinder (Narishige Model EG-4). This instrument bevels the pipette to increase opening diameter while retaining the small size of tip. 12 Eppendorf microinjector (Model ECET 5242). This instrument is equipped with switch controls that provide injection pressure, holding pressure, and negative pressure (vacuum). The vacuum mode is used to fill micropipettes. The instrument operates using compressed gas (air or nitrogen). 13 (a) Complete video microscope microdissection apparatus.

Remove aggregates by gently drawing up solution in a Pasteur pipette. 9. Immediately centrifuge at 150 g for 2 minutes. Suction off supernatant. 10. Repeat Steps 8 and 9. 11. 5 ml 3 : 1 methanol : acetic acid and place on ice. 12. Immediately drop 6 - 7 drops of fixed cells onto large (35 x 50 mm), cold, wet coverslips using a Pasteur pipette. The coverslips should be kept at - 7 0 ° C for a few hours before use. Immediately before use, dip coverslips into ice-cold distilled water. Drop cells from a height of approximately 2 - 3 feet (arm's length) onto a slide held at a 30° angle.

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