Crushed ice samples

Crushed ice samples. Figure 2 shows the dielectric properties of samples consisting of packed crushed ice particles, which were prepared by crushing single crystal... 

The tissue of interest should be removed from the animals using equipment that has been treated to ensure that it is RNase-free. The surface hair of the animals can be soaked in 70% ethanol to minimize the inclusion of hair or dander in the isolated tissues. A sterile disposable Petri plate, placed on top of a bed of crushed ice, is a suitable RNase-free surface for microdissection. Immediately after isolation from the animal, individual samples of tissue can be flash frozen by immersion in liquid nitrogen and stored in cryovials in liquid nitrogen or at -70°C until all samples from an experimental set have been obtained. RNA extraction and preparation of single-stranded cDNA can then be performed simultaneously on all samples. This will minimize differences between RNA populations that result from differences in sample preparation. 

The sample containing up to 200 pg of protein is filled up to 1.0 ml with ddH20. After that, 2.0 ml of Soln. A are added. After mixing, the samples are put into crushed ice for 10 min. Centrifuge the samples thereafter in a refrigerated centrifuge at 4 °C with 4000 x g for 5 min. 

The smallest vertebrates are homogenized whole and most laiger animals can be dissected and different tissues removed for storage in 1.5-ml Eppendorf microtubes at — 70° until homogenized. Heart, kidneys, or liver are sufficient to score more than 20 proteins testes, spleen, brain, and muscle can be sampled for tissue-specific enzymes. Additionally, blood, muscle, and/or saliva from most vertebrates can be conveniently sampled without killing the animal. During all sample preparation steps, keep tubes on crushed ice. 

Using a hypodermic syringe, withdraw a sample of about 5 uiL of each solution as soon as possible after it is made up. Chill these samples by placing them in test tubes set in crushed ice, then put them aside for absorbance measurements (to be made as soon as conveniently possible). These measurements will provide a value for sd in case it is needed later on. 

Figure 10-2. Electric blenders capable of handling sample volumes of 10 ml (a) to 4 liters (b). The instrument in (a) is equipped with a dish which may be filled with crushed ice in order to...

Figure 2 For packed samples of crushed ice particles, (a) Temperature dependence of the dielectric relaxation time rand (h) Cole-Cole plots of the sample (after 400 h of annealing at -1 °C, 2-4 mm particle size, 536 kg/m packed density) at -10 °C. The dielectric dispersion is of the Davidson-Cole type (a=0.97, f=0.85).

Sample preparation Keep tubes in crushed ice except when being processed throughout this procedure. 2 mL Plasma + 100 p,L 10 p.g/mL sulfanilamide in MeCN + 8 mL diethyl ether, vortex for 2 min, centrifuge at 4°. Remove ether layer and add it to 1 mL 100 mM NaOH, vortex for 2 min, centrifuge at 4°. Remove aqueous layer and add it to 1 mL 100 mM HCl and 500 j,L 50 mM pH 7.4 sodium phosphate, add 8 mL ether, vortex for 2 min, centrifuge at 4°. Evaporate ether layer to dryness, reconstitute in 200 p,L mobile phase, iiyect 50 xL aliquot. 

Sample preparation Plasma. 750 pL Plasma -I- 50 pL 320 pg/mL IS -I- 25 pL 85% phosphoric acid + 750 pL EtOH isopropanol 75 25, vortex for 10 s, let stand on crushed ice for 30 min, centrifuge at 1000 g for 15 min. Remove a 400 pL aliquot of the supernatant and add it to 400 pL 10 mM pH 5 phosphate buffer, vortex for 10 s, iqject a 250 pL aliquot onto column A with mobile phase A and elute to waste. After 4 min backflush column A with mobile phase A to waste, after 1 min backilush the contents of column A onto column B with mobile phase B, after 10 min remove column A from the circuit, elute... 

After addition of suitable internal standards the fractions are concentrated by rotary evaporation to > 1 mL. The rotary evaporator should be fitted with a solvent trap as shown in Fig. 18-2 to prevent refluxing solvent to flow back into the attached flask. The rotary evaporator air inlet should either be connected to a tank of compressed nitrogen or be fitted with a 5 A molecular sieve trap. The concentrated samples are transferred with a 100-250 mL syringe into glass ampoules as shown in Fig. 18-3 which are flushed with nitrogen and flame-sealed while being cooled to well below room temperature with a mixture of crushed ice cubes and table salt. 

For subsampling from a water bottle, the syringes are first rinsed three times with the sample water and then filled carefully so as not to leave any air bubbles inside. The syringes are stored immersed in seawater to prevent any possible exchange between the water inside and ambient air. The water in a bucket, taken from the surplus water in the samplers, is replaced regularly. Also, the syringes should be kept cold before analysis, preferably with crushed ice, or in a cold store. The reason is that seawater of low temperature inevitably creates small gas bubbles inside the syringes when the temperature increases. 

The four absorption tubes were maintained at -72 C in a dry ice-methanol bath. Water was precondensed in U-tubes at -15 C (sodium chloride in crushed ice). The flow through the traps averaged 70 ml min" over periods from 30 min (exhaust), to 18 h. Each trap was kept in dry ice until it was attached to a 4-way valve installed between the carrier gas inlet and the injection port of the gas chromatograph. The trap was immersed in boiling water. After 1 min, the volatilized fraction of the sample was introduced to the g.c. column by diverting the carrier gas through the 4-way valve. 

Liquid Samples. Liquid samples such as plasma or milk are shell-frozen in the lyophilizer flasks using a mixture of crushed dry ice and 2-propanol. They are then dehydrated in the same way as the solid samples. The resulting material, which is brittle and spongy and easily broken up, is then pressed into cans and sealed. No preservative is added to the dried materials they can be stored indefinitely at room temperature. Approximately 2 liters of fresh milk can be dried and compressed into one can. 

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