careful blending was attempted with constant mixing. ph of the mixture was then raised to 5.5 with 1n Naoh and the mixture was left at room temperature (220c) overnight (Ph5.5 is the isoelectric purpose of Igg/ F(ab)2). Centrifugation of solvent protein: The above mixture was spilled in the axis containers in equivalent sums and centrifuged at 2500rpm for 30 minutes. The supernatant was disposed of and the encourages were gathered. Dialyses of the accelerated
Plasmid DNA was precipitated from the supernatant by adding 0.7 volumes of isopropanol and centrifugation at 13,000rpm for 30min and washed with 70% ethanol. The pellet was air dried, resuspended in TE buffer (pH 8.0) with RNase 10μg/ml and allowed to dissolve completely. The isolated plasmid DNA was electrophoresed on 1 % agarose gel (containing Ethidium Bromide) at 75 volts and visualized under UV light in order to check its
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.
The mixtures was filtered with Whatman #1 filter paper and the filtrate was used to repeat the extraction for another two times. Then, the methanol solvent in mixtures was removed using the Rotary Evaporator (EYELA, N1100) at 140° hPa; 60°C; speed 5. The distilled water and excessive methanol were removed with Freeze Dryer (GENEVAC LTO, EZ 2.3 ELITE) for a week. The crude obtained were weighed and stored at -20°C until use. 1.2 Partitioning method
Agitation speed was maintained at 350 rpm which helps in complete removal of methylene chloride. Microspheres were then collected, filtered, washed three times with distilled water and stored under reduced pressure, overnight in a desiccator. Additionally, optimization of the process was done by selecting suitable stirring device element i.e. magnetic stirrer vs mechanical stirrer in order to improve the shape and yield of microspheres. A paired t- test was applied for final selection of the stirring element based on 95% confidence
The reaction mixture was poured into ice cold water acidify with 1% HCl and precipitates were collected, filtered and dried and recrystalized with ethanol. 4.2.2 General procedure for synthesis of flavones: 50mg of chalcone was taken in round bottom flask. After that 15-20 ml of DMSO was added and mixed properly, 5-6 granules of iodine were added and reflux the reaction mixture up to 3-6 hours and kept for overnight. Then reaction mixture was poured into ice water and the precipitates were neutralized with sodium thiosulphate, to remove the unreacted iodine, washed with water, filtered, dried and recrystalized with
We started by putting 100 mL of water into a coffee cup calorimeter (a polystyrene cup inside another polystyrene cup as an insulator), a magnetic stir bar was added and using the program LabProTM the initial temperature was recorded. The 5.7 grams of solid Mg(OH)2 were added and data was recorded until the temperature remained stable for one minute. Next, we recorded the initial temperature of the slurry, then we added 14.1
After which the digestions were examined by gel electrophoresis. The samples were run on a 50 mL 0.9% (w/v) agarose gel in 1X TAE buffer at 100 V until the leading track dye traveled 2/3 the distance of the gel. The gel was then soaked in GelRed for 20 minutes and examined under UV light. To prepare the digestions 10 μL of each digestion was mixed with 2 μL of 6X track dye in a micro centrifuge tube. 12 μL of 1 kb DNA ladder and each digestion was run on the gel.
We inoculated them with our stain and incubated them on shaker for 24 hours. After 24 hours incubation, we took O.D at 600nm and plotted a graph. To check effect of different temperatures on rhizospheric nitrogen bacterial growth: We took 6 flasks (100ml), and added 20ml L-broth in each flask and autoclaved it. We inoculated them with our stain. We selected different temperatures 4°C, 15°C, 37°C.
harveyi by biochemical test one colony of V. harveyi was taken into 1.5 ml eppendorf tube by isolating loop into 0.9ml peptone water and prepared stock solution of V. harveyi. The stock solution was diluted (tenfold dilution) with peptone water (James and Hirsch 1960) and prepared test solution of V. harveyi. Then 0.5 ml isolated Lactobacillus spp. probiotic solution was separately mixed with 0.5ml of test solution (V. harveyi). One ml suitable dilution of the mixer solution was inoculated in TCBS agar media after at subsequent intervals of 4 hour up to 12hour.