Cell Biology. A Laboratory Handbook, Volume 3 by Julio E. Celis

By Julio E. Celis

A pragmatic, inclusive laboratory significant other, this guide offers telephone biologists and different lifestyles scientists with the main updated directions for uncomplicated and complicated mobile organic thoughts. approximately 2 hundred articles by way of top scientists from all over the world are very easily prepared into 1 five elements in 3 separate volumes. 1000's of good-looking diagrams and images plus 33 colour plates improve the layout and value of this new paintings. Uniform type and enhancing through an skilled phone biology laboratory path teacher and examine staff chief make certain caliber and accessibility to a wide and growing to be foreign viewers of mobile, developmental, and molecular biologists, plus others who want those tools of their laboratory study

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1 0 . Purify mRNA by passing total RNA through oligo(dT) spin column. 4. Reextraction of RNA from Microinjected Cells for Northern Blot Analysis Solutions 1 . 0 with HCl, and bring the volume to 25 ml. 2. Proteinase K: Dissolve 1 mg proteinase K in 100 µl DEPC water. The solution cannot be stored. 3. tRNA: Dissolve 10 mg tRNA in 1 ml DEPC water. Store at -20°C. 4. Lysis buffer: Prepare immediately before use. 5 µl of tRNA, and bring the volume to 500 µl with DEPC water. 5. 5 g of NaOAc in sterile, DEPC-treated water, adjust pH to 6 with acetic acid, and bring to a total volume of 1 00 ml.

The pipette Puller type P-8 7 i s from Sutter Instruments. The microin­ j ector (Model 5242), prepulled and sterile ready-to-use micropipettes ( Cat. No. 008), and microloaders ( Cat. No. 222) are from Eppendorf. The automated injection system (AIS) used was developed at EMBL (Ansorge and Pepperkok, 1 9 8 8 ) (see Fig. lA). The commercial version (see Fig. l B ) is distributed by Zeiss. An inverted microscope (Axiovert 1 0) with long-distance phase-contrast optics (magnifications: 4 5 X -320 X ) is used.

9. Lower the micropipette carefully until the tip comes into focus. Both cells and tip are on the same focal plane. 10. Use the computer mouse to mark the tip position (AIS menu program "Mark Tip"). 11. Proceed directly with automated microinjection. C. AUTOMATED M ICROINJECTION For the following, the micropipette should be centered in the field of view as described in Section B. The pressure applied to the micropipette remains constant during automated injection. It is adjusted at the beginning of the session with the Computer-Automated Microinjection of Macromolecules into Living Cells 25 microinjector according to the cell type and injection sample used [see also the Zeiss AIS Operating Instructions ( 1 989)].

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