One unit (U) of glucoamylase is defined as the amount that liberates 1 µmol of reducing sugar as glucose/ml/min under the assay condition. One ml of the diluted enzyme extract was added to 1.0 ml of 5% soluble starch solution prepared in acetate buffer (pH 4.8). The enzyme substrate mixture was incubated at 60 0 C for one hour. Then 2 ml of Dinitrosalicylic acid reagent (DNS) was added to each test tube. The test tubes were placed in boiling water for 5 minutes and cooled to room temperature.
Transformation #1 corresponds to the tube to which ligation #1 was added, and so on for each of the four transformations. After ligations were added each solution was chilled on ice for 5 minutes then heat shocked at 42° C for 5 minutes, then again chilled on ice for 2 minutes. To each tube was then added 80 μL room temperature Luria Broth. After which the solutions were incubated at 37° C for 45 minutes. Each solution was then plated on an LB/kanamycin/IPTG plate.
The P2 tube was then placed in the refrigerated centrifuge at a speed of 15,000g for 30-60 minutes at 4°C. 1ml of the homogenisation buffer was added to the P1 pellet and was vortexed to resuspend it. The supernatant was then removed from the P2 tube and placed into a micro tube labelled ‘S’. 1 ml of the homogenisation buffer was added to the P2 pellet and placed on ice. The pellet was then resuspended again by adding small quantity of glass beads and it was vortexed vigorously until the pellet has disappeared from the bottom of the
The incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.
The aqueous extract was prepared by dissolving 1g of dry extract with 20 ml of sterilized distilled water, so the final concentration of extract would be 0.05 g/ml, from this solution other concentration were prepared (0.1-0.2) g/ml. the solutions were shaken for 30 min. The extract was centrifuged (30,000 rpm; 15 min) and the supernatant was Separated. To hydroalcoholic extract, 80 g of the powder was extracted with aqueous methanol (75%). The other two concentrations were prepared from soaking sixfold aqueous methanol (75%) with different amounts of powder.
The mixture was sonicated for 5 minutes and then followed by a degassed and purged process with nitrogen gas for 15 minutes in the ice bath. The opening of the conical flask was sealed with the aluminium foil and paraffin to prevent oxygen from entering the flask along with the heating process. The conical flask was then emerged in a water bath for 12 hours. The temperature is monitored to be maintained at 60oC for 8 hours to allow the initiation of the reaction and increased to 85oC for the remaining 4 hours. After drying overnight, a rigid structure of the light brown polymer was obtained.
The optimized ratios of the polymers were calculated by using 22 factorial design. (Table 1). Ethyl cellulose (2gm) was dissolved in 20 ml of methylene chloride2. The polymer phase of ethyl cellulose was then added to 250 ml of 0.25% w/v methylcellulose aqueous solution (over night dispersion). Agitation speed was maintained at 350 rpm which helps in complete removal of methylene chloride.
in the first step benzoic acid was reacted with excess of thionyl chloride using acetonitrile as a solvent and keeping the mixture on an ice bath for 3-4 hours (labeled as reaction mixture a) In the second step gemcitabine hydrochloride along with 3eq tri-ethyl amine and using ethanol again as a solvent was stirred for 15-20 minutes without ice-bath. next with a poisterizing tube the reaction mixture a was drop wise added to reaction mixture b yielding a third and final, reaction mixture c giving off white fumes of socl2. it is stirred for 19 hours and 15minutes at 80c and colour changes to light yellow The preparation of benzoyl chloride from benzoic acid using thionyl chloride at 0’c is an in-situ preparation procedure: FILTERATION: Evaporate reaction mixture and dissolved in hexane and then filter it. The best TLC system for filterate is ethyl acetate : hexane , 4.5:0.5 3.3.2 PROCESS 2 FIGURE 3.5ACETYL DERIVATIVE PROCEDURE In 75mg of gemzar, 2ml ethanol is added and then solubility is checked. After 5 minutes add 0.1 ml (5 drops) DMF, then add 0.104ml Et3N and add 0.036 ml acetyl chloride and stirr it for 17 hours r at 47C
CHAPTER III RESEARCH METHOLOGY 1.0 Plant extraction of Physalis minima 1.1 Preparation of fresh sample extract Physalis minima L. at maturity state was harvested from Kepala Batas, identified and washed with a distilled water. The dried specimen was submitted to the USM Herbarium for future reference. About 200 g weight of leaves were blended finely with 300 mL distilled water using a blender until become a mixture with a small size of leaves. The water mixture of leaves were macerated with 1200 mL methanol and shaken continuously at 250 rpm for 24 hours. The mixtures was filtered with Whatman #1 filter paper and the filtrate was used to repeat the extraction for another two times.
To 200 µl sodium nitroprusside (5Mm), 800 µl extracts (0.1-1 mg/ml) dissolved in PBS (25 mM, pH 7.4)was added.The mixture was incubated for 2.5 hrs. at 37ºC under normal light followed by incubation in dark for 20 min. 600 µl Griess reagent was added and incubated for 40 min. at room temperature and absorbance was measured at 540 nm against a suitable blank (2ml H2O and 0.6 ml Griess reagent). Control (1.6 ml H2O, 400µl SNP and 600µl Griess reagent) was prepared and percent of inhibition was calculated by using this