Pipetting Pasteur pipettes

Use a long Pasteur pipette to layer 2.2 ml of the 10% sucrose solution in a Beckman polyallomer tube (14 x 89 mm, 331372). Then, underlay 2.2 ml of the 20% sucrose solution by inserting the tip of the pipette to the bottom of the tube and slowly pipetting the 20% sucrose solution under the 10% solution. Underlay the 30, 40, and 50% sucrose solutions in the same manner. Cover the tube with aluminum foil and store overnight at 4° to establish a linear gradient. 

Pipet 4 ml of the hot molten Soln. A onto the covered slides laying on a horizontally leveled table. After gelation, punch wells using a stamp as shown in Fig. 4.1. Suck out carefully residual material from the wells using a Pasteur pipette connected to a pump. 

Pipetting is one of the most frequent operations carried out in the biochemistry laboratory. Simple Pasteur pipettes are intended for the non-quantitive transfer of liq-... 

Figure 2-2. Class pipettes. Pasteur pipettes (top) are designed for transferring small quantities of liquid (< 2 ml), for example, from a test tube into a spectrophotometer cuvette. Bulb (middle) or graduated (bottom) pipettes are designed for pipetting specific fixed and variable amounts of liquid respectively.

The required solvent layer is then removed using a Pasteur pipette. Three or four extractions are usually sufficient to recover the majority of the material. This technique is particularly straightforward for diethyl ether extractions of aqueous solutions in which the edier layer is required, since it can readily be pipetted from the top of the aqueous layer (Fig. 12.1). If you require the lower solvent layer, this can be recovered either by pipetting away the top layer, or by pipetting from the bottom of the vial. Because efficient separation of the two phases is required, it is preferable to use a tall, thin vial rather than a short, fat one. A second vial containing drying agent can then be used to dry the extracts. 

Using a Bounce homogenizer with a lose-fitting pestle, thoroughly homogenize tissue in Homo buffer continue until the homogenate can be pipetted with a Pasteur pipette. About 1ml/100 flies. 

Polymerization is initiated by adding 20 ml 1.4% ammonium persulfate solution and mixing well. This should be done immediately before use. The monomer mixture is carefully pipetted into each of the spaces between the glass plates and the sides of the Perspex box. It should rise to a height of 6 cm above the top of the gel which is already there. A thin wire is used to remove any air bubbles on the walls. Finally, a Pasteur pipette is used to add a 2-3 mm layer of water on top of the monomer, avoiding mixing at the interface as far as possible. Polymerization is complete after 30 minutes. 

Estimate the volume recovered, add 5 /rl of cytochalasin B per milliliter of cytoplasm, and mix gently by pipetting with a Pasteur pipette. Then recentrifuge at 15,000 rpm for a further 15 min at 4°C. 

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