Buffers and mixes

10 x Restriction buffer 500mM-NaCl, 66mM-MgCl2, 66mM-2-mercaptoethanol, 66 mM-Tris HC1 pH 7.4. 

Marker dye mixture Mix equal volumes of 1% Xylene Cyanol F.F. (blue), 2% Orange G (yellow) and 1% Acid Fuchsin (pink) (all from George T. Gurr, Ltd., London.) 

The plus and minus method represented a major breakthrough in the development of primed synthesis methods for the rapid determination of DNA sequences. 

Like any method however, there are certain limitations and 

Taken all in all the plus and minus method is a powerful technique. However, it does have its limitations and the more recent developments using the dideoxynucleoside triphosphates as specific chain inhibitors have much improved both the speed and accuracy of the method and will usually be the method of choice for primed synthesis sequencing. This is described in the next chapter. 

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Add 1-10 mg of a protein or antibody containing an available thiol group to the particle suspension in coupling buffer and mix to dissolve. Alternatively, add the protein to the particle suspension in an amount equal to 1-10 X molar excess over the calculated... 

Substrate/buffer solution. Dissolve 6.8 mg 4-methylumbelliferyl-/f-D-glucopyran-oside (MW 338.3) in 3.6 ml of reaction buffer to a final concentration of 5.6 mM. Add 0.4 ml of taurocholate in reaction buffer and mix. The final substrate concentration is 5 mM. 

Add 66 pL of NBT solution to a plastic tube containing 15 mL of alkaline phosphatase buffer, and mix it by gentle swirling. Then add 50 pL of BCIP (toluidine salt) solution, and mix it again... 

A final example of totally automated HPLC (although it isn t for pesticides) will demonstrate how many different unit operations can be done in a single system to take the tedium out of repetitive analyses. The drug analyzer depicted in Figure 14 was designed to determine therapeutic levels of theophylline in human serum (8). The sampler (in the center) aspirates 50 uL of serum into the analytical cartridge, then to the EDM, and finally to the LC module. The following series of operations takes place at the rate of 20 samples per hour without operator intervention unmeasured, untreated sample is aspirated, diluted with buffer, and mixed with an internal standard the system then precipitates the protein, removes the particulates, extracts the analyte (and internal standard) into... 

Add acrylamide and APS to one-half of the total volume of gel buffer and mix well with constant stirring at room temperature. 

NAD Solution Transfer 40 mg of nicotinamide adenine dinucleotide (B-NAD, Grade III-C, from Sigma Chemical Co.) into a glass vial, dissolve it in 10.0 mL of Phosphate Buffer, and mix. 

Test Preparation Transfer about 2 g of sample, accurately weighed, into a 100-mL volumetric flask, dissolve it in 50 mL of Phosphate Buffer, dilute to volume with Phosphate Buffer, and mix. Insert a stopper into the flask, heat at 60° for 1 h, and cool to room temperature. 

For sample blanks, transfer, in sequence, 1.0 mL of each sample to separate test tubes, add 5.0 mL of Borax Buffer, and mix. Add 2.0 mL of Substrate Solution to each tube, and mix. 

Test Preparation Prepare a solution from the enzyme preparation sample so that 1 mL of the final dilution will contain between 0.01 and 0.02 (3-glucanase units. Weigh the sample, transfer it into a volumetric flask of appropriate size, dilute to volume with Phosphate Buffer, and mix. 

Glucose Substrate Dissolve 539 g of anhydrous glucose and 1.0 g of magnesium sulfate (MgS04-7H20) in 700 mL of water or the manufacturer s recommended buffer, previously heated to 50° to 60°. Cool the solution to room temperature, and adjust the pH as specified by the enzyme manufacturer. Transfer the solution to a 1000-mL volumetric flask, dilute to volume with water or the specified buffer, and mix. Transfer to a vacuum flask, and de-aerate for 30 min. 

ONPG Substrate Dissolve 250.0 mg ONPG (use lot currently in use) in about 80 mL of P-E-M Buffer. Transfer the solution to a 100-mL volumetric flask, dilute to volume with P-E-M Buffer, and mix. Prepare, at most, 2 h before incubation. 

Standard o-Nitrophenol Solution Transfer 139.0 mg of o-nitrophenol into a 1000-mL volumetric flask, dissolve in 10 mL of 96% ethanol, dilute to volume with water, and mix. Pipet 2-, 4-, 6-, 8-, 10-, 12-, and 14-mL portions of this solution into a series of 100-mL volumetric flasks, add 25 mL of Sodium Carbonate Solution to each, dilute each to volume with P-E-M Buffer, and mix. The dilutions contain, respectively, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14 pmol/ mL of o-nitrophenol. 

Substrate Solution Dissolve 8.40 g of sodium phytate dec-ahydrate (C6IIgC PgNa -101120) (Sigma Chemical Co.) in 900 mL of Acetate Buffer. Adjust the pH to 5.50 0.05 at 37.0° 0.1° by adding 4 M acetic acid. Cool to ambient temperature. Quantitatively transfer the mixture to a 1000-mL volumetric flask, dilute to volume with Acetate Buffer, and mix. Prepare fresh daily. 

Phytase Reference Solutions, Procedure 1 Weigh an amount of Phytase Reference Preparation corresponding with 20,000 phytase units accurately to within 1 mg in duplicate in 200-mL volumetric flasks. Dissolve in and dilute to volume with Acetate Buffer, and mix. Dilute with Acetate Buffer to obtain dilutions containing approximately 0.01, 0.02, 0.04, 0.06, and 0.08 phytase units per 2.0 mL of the final dilution. 

Sample Preparation Using Glycine-Hydrochloric Acid Buffer, prepare a solution of the sample enzyme preparation so that 2 mL of the final dilution will give a corrected absorbance of enzyme incubation filtrate at 275 nm (AA, as defined in the Procedure) between 0.200 and 0.500. Weigh the enzyme preparation, quantitatively transfer it to a glass mortar, and triturate with Glycine-Hydrochloric Acid Buffer. Quantitatively transfer the mixture to an appropriately sized volumetric flask, dilute to volume with Glycine-Hydrochloric Acid Buffer, and mix. 

To one tube at a time, rapidly add 45 mL of ammonium chloride lysis buffer and mix immediately. 

Phenol Red. The first publications of an automated procedure for the measurement of cholinesterase inhibitors are those of Winter 14) and Winter and Ferrari (15). The method employed an Autoanalyzer instrumental system wherein the extracts containing the insecticide were incubated with a standard cholinesterase solution at 37 °G. A continuous sample from the incubation bath is buffered and mixed with acetylcholine iodide. After a second incubation, the acetic acid released by the action of the uninhibited cholinesterase is measured colorimetrically, using phenol red as the indicator. More recently, Fischl et al. 16) reported a method for rapid detection of organic phosphate pesticides in serum. Strips of filter paper were impregnated with a buffered acetylcholine substrate solution containing phenol red as an indicator. When no inhibition is present, the acid released from the action of cholinesterase turns the paper yellow. When cholinesterase has been inhibited, the paper turns pink-to-violet. 

In the direct UV method, compounds are dissolved in DMSO stock solution at 10 mg/mL. A small volume is added to an aqueous buffer and mixed. If the target concentration exceeds the solubility of the compound, the insoluble material will precipitate. The solution is allowed to settle for certain period of time (e.g. overnight) and is then filtered to remove the precipitate. The concentration of the supernatant is determined by using a UV plate reader and the solubility is derived against a single point standard (Avdeef, 2001). 

The relatively slow kinetics of complexatlon has been proposed as a cause of the generally low efficiencies of boronate stationary phase systems (19). The plate numbers of interacting solutes in these studies are about 90% of otherwise observed and assyraetry is unchanged. The latter is unusual since previous work showed tailing ascribed to the presence of a "non-linear Isotherm" (20). Perhaps the combination of surface buffering and mixed bonded phase effect creates a more favorable, more uniform sorption environment. 

Serum pseudo-cholinesterase (referred to hereafter as serum cholinesterase) activities were determined from a five minute reaction period at room tenperature with a modification of the method of EUman et 2Q. ( ). Briefly, a 10 pi 2d.iguot of serum ves added to both a reference and sample cuvette ocxitaining 3.0 ml of 5,5-dithiobis-(2-nltrobenzoate) (DDO buffer (0.25 mM in ph 8.0, 0.1 M sodium phos te buffer) and mixed with a micro-stirring rod. Die reaction ves steurted in the sample cuvette by the addition of 20 pi of aoetylthiochollne iodide (78 nM) and stirred. The reference cuvette received 20 pi of distilled water in place of substrate. Absorbance was measured at 412 im over a five minute period and the rate calculated from the slope of the derived curve. 

Make up protein samples [80-100 pg of radioactively labeled proteins, 3 x 50 pg of DIGE-labeled proteins (see Note 5), or 350-600 pg of unlabeled proteins for Colloidal Coomassie staining) (see Note 6)] to 360 pL with 8 M urea/2 M thiourea. Subsequently, add 40 pL of lOx rehydration buffer and mix the solution by shaking at room temperature for 30 min. Centrifuge the rehydration mix for 5 min at 21,000 x g (20 °C) to remove insoluble proteins. 

Lab-on-a-Chip Devices for Sample Extractions, Fig. 3 An integrated cell lysis and DNA extraction device. Parts (a-e) show the sequence of steps involved in introducing the cell sample and lysis buffer and mixing... 

For each 5 pi RNA samples, add 5 pi of RNA loading buffer, and mixed by gentle flicking. 

Discard supernatant. Wash protein-bound beads with 750 pL washing buffer and mix gently. 

Block any imreacted DMP sites resuspending the gel in an equal volume of blocking buffer and mixing by inversion for 10 min at room temperature. 

Into three 1.5-mL microtubes, pipet 10 pL of the diluted labeling reaction mixture (see step 5 in Subheading 3.1.) Add 490 pL of ice-cold assay buffer and mix by vortexing To this, add 500 iL of ice-cold 20% TCA and mix by vortexing... 

Resuspend in 10 pL of electrophoresis buffer and mix with 10 pg of each standard phosphoamino acid. 

Add 1.0 ml of hydroxylamine solution from an automatic pipette, followed by 2.0 ml of sodium acetate buffer and mix the contents of the cylinder by shaking (Note c). 

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