Centrifuge tabletop

Electron microscope Knife breaker Light-tight darkroom Tabletop centrifuge Ultramicrotome Water bath, 45°C Wire loop oftungsten or platinum... 

Equipment PCR machine, scintillation counter, tabletop centrifuge, temperature-controlled water baths, equipment for horizontal and vertical electrophoresis, UV-illuminator, phosphor imager, automatic DNA sequencer, vacuum dot-blot manifold (Schleicher and Schuell). PCR 0.5 ml hot-start mbes, aerosol resistant pipette rips, autoclaved Eppendorf tubes (all from Fischer Scientific, Brightwaters, NY) and glassware, diethyl pyrocarbonate (DEPC, Sigma)-treated solutions. 

For precipitation of DNA and RNA, add 10% of the volume sodium acetate, pH 7 or 5.2, respectively. Add four parts of ice-cold 98% ethanol and mix well. Add 1 pi of linear acrylamide (3%). Incubate for 2 h at -20°C and centrifuge at 12,000 for 30 min in a tabletop centrifuge. Carefully remove the supernatant—the DNA or RNA should be visible against the light as one or several small shiny specks, usually attached to the tube, but they may be loose—and wash the pellet with 200 pi 80% ethanol. Remove the supernatant, and dry the DNA or RNA (3 min in a speed vacuum concentrator) until all visible liquid has gone, but avoid overdrying of the pellet because the DNA or RNA will then be difficult to redissolve. Dissolve the DNA in 100 pi dd H O. 

Remove excess PBS and immediately apply 50 pi double-FAM LNA (see Note 2) probe solution and gently shield with cover glass (see Note 3). The probe solution is prepared as follows denature LNA probe and dilute the probe in Exiqon ISH buffer. For example, for 2 ml hybridization mix containing 20 nM double-FAM-labeled miR-21 LNA probe (from 25 pM probe stock), transfer 4 pi into the bottom of a 2 ml nonstick RNase-free tube and place the tube at 90°C for 4 min. Spin down shortly using a tabletop centrifuge, and immediately... 

Extract the chlorophyll from the chloroplasts by mixing, in a conical centrifuge tube, 0.05 mL of well-mixed chloroplast suspension with 9.9 mL of 80% acetone in water. Spin in a tabletop centrifuge for 10 minutes. Transfer the supernatant to a glass cuvette and read the absorbance at 652 nm using 80% acetone in water as reference. Calculate the concentration of chlorophyll in the chloroplast suspension using Equation E9.3. 

Tabletop centrifuge, rotor, and 50- to 500-ml tubes, or sintered glass funnel Whatman No. 1 filter paper in a Buchner funnel Separatory funnel Rotary evaporator...

Centrifuge the tubes for 5 min at 1000 x g, room temperature, in a tabletop centrifuge to separate the organic and aqueous phases. 

N-EVAP model 112 nitrogen evaporator (Organomation) with moisture trap in line from N2 source 50°C heating block Test tube shaker Tabletop centrifuge GC sample vials with Teflon caps... 

Polytron PT-3100 homogenizer equipped with a PT-DA 3012/2EC aggregate (probe) or equivalent Whatman no. 40 filter paper 15- and 50-ml test tubes with Teflon-lined screw cap Test tube shaker Tabletop centrifuge Vacuum aspirator... 

Tabletop centrifuge and appropriate swinging bucket rotor Vacuum filtering apparatus and Whatman filter no. 42 paper 30- to 50-ml tube with cap Shaking water bath or ultrasonic bath, 60°C...

Explosion-proof tabletop centrifuge or a cooling tabletop centrifuge UV-VIS spectrophotometer 1-cm-path-length cuvette 25- or 50-ml separatory funnel Water bath set below 35°C (optional)... 

Centrifuge 5 min at 300 to 500 x g in an explosion-proof tabletop centrifuge at room temperature. 

Pellet cells (200-500 pL) by centrifugation in a tabletop centrifuge for 2min. 

Pool the 12 reactions and extract once with 1 volume of phenol/chloroform and once with 1 volume of chloroform. Precipitate the DNA with 1/10 vol of 7 M ammonium acetate and 3 vol of 96% (v/v) ethanol. Incubate for at least 30 min at —20 °C, then centrifuge in a tabletop centrifuge for 15-30 min. 

The reaction is assembled in 15 ml conical tubes, and then is incubated for 2-3 h at 37 °C. If no inorganic pyrophosphatase is added, the solution will turn cloudy over time as pyrophosphate is released. If pyrophosphatase is not used, then at the completion of the reaction, this precipitate must be pelleted in a tabletop clinical centrifuge for 10 min and the supernatant immediately removed. 

Preparative centrifuge Clinical tabletop centrifuge (low speed) Spectrophotometer to read absorbance at 500 and 660 nm with cuvettes 100°C oven... 

Separate the methanobwater phase (top) from the chloroform phase (bottom) in each tube by centrifugation at low speed (—500 X g, 5 min) in a tabletop centrifuge. You will see a small disk of white precipitate (denatured protein) at the interface between these two phases. If this disk extends down too far into the chloroform layer (more than halfway into the chloroform phase), add 0.1 ml of 0.1 M HC1, mix, and repeat the last low-speed centrifugation step. 

Clinical tabletop centrifuge (low speed)—At least one required. 

Tabletop centrifuge with aerosol resistant containers accomodating 15-mL centrifuge tubes. 

Pellet in tabletop centrifuge at 2300g for 10 min at 4 C. Resuspend pellet in 16 mL of buffer 2 (containing protease inhibitors) at room temperature. Rock gently at room temperature for 10 min. 

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