coli transformants was isolated by alkaline lysis method (Sambrook et. al., 1989). Clones obtained on plates were inoculated in 5ml LB broth containing appropriate antibiotic and incubated overnight at 37°C under shaking conditions. The cells were harvested by centrifugation at 7000rpm for 5min and resuspended in 200 μl of GTE (Glucose-tris-EDTA) buffer. For lysis two volumes i.e.
All reaction tubes, enzyme stock, and the two tubes with water were warmed in a 37 °C water bath for ten minutes. After heating, the reactions were started by adding 250 µL of the enzyme solution into each of the A and B tubes and 250 µL of the deionized water to the C tube. The tubes were kept in the water bath for 15 minutes. After the 15 minutes, 750 µL of 0.20 M NaOH was added to each tube to stop the
After 8 days incubation, 5ml sterile distilled water was added on to the agar using aseptic techniques. The top of the agar was scrapped with a sterile hockey stick glass rod so that the spores was suspended into the solution. The concentration of the spore was determined by using hemocytometer. A proper dilution was carried out to get a spore concentration of 2 x 106. Each isolates was inoculated into 50ml cellulase enzyme production broth medium with inoculum size of 2 x 106 spore concentration.
primarily each isolate was cultured in broth medium and incubated on 37 C. overnight. Suspensions of any isolates were prepared that turbidity equal to 0.5 McFarland standards(OD=0.1). Each bacterium had been swabbed with those suspensions on Muller Hinton agar medium. Four wells were created (6mm in diameter and the distance of 25cm from each other) with sterile cork borer. Each well was poured with 100 μl clear supernatant liquid of any concentration.
Repeat the experiment. The cola drinks were titrated using the following method: Prepare the beverage in a 250ml volumetric flask. Use a funnel to facilitate the process. Place the beaker on a hot plate so that it boils and place a watch glass on top to prevent the carbon dioxide from the atmosphere getting dissolved in the cola. Once the cola starts to boil, continue to boil it for another 10 minutes so that the carbon dioxide is removed.
The DEAE CL-6b gel was washed twice with 0.5m Hcl, twice with 0.5m Naoh and twice with PB ph6.0 before utilization. For DEAE 1ml gel every 1ml serum was utilized. In the wake of blending for 1 hour at 200c the suspension was centrifuged at 4500g for 25 minutes. The supernatant was thought by Ammonium sulfate precipitation and pellet was broken up in refined water and the ensuing arrangement
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.
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.
The oxidizing agent (in terms of different proportions) was first dissolved in 10 ml of distilled water and then added drop wise to the PVA + pyrrole mixture. This composite solution was gently stirred for 5 hours. A homogeneous black colored solution was obtained. Films of this solution were prepared by pouring a certain small portion on to a flat glass Petri dish or polypropylene surface. The thickness of the film was controlled by the volume of the solution added.
1- Extraction method No. 1: Fifty grams of powdered aerial parts of portulaca oleracea were hydrolyzed by using reflux for 9 hr. with 300 ml of 2N hydrochloric acid then the extract cool at room temperature ,filter and wash the residue with 2N of ammonia solution. The residue dried overnight at 60ºC ,the final step involve the extraction of the residue with 250 ml of chloroform by using soxhlet ,the final extract cool at room temperature ,then filter and evaporate to dryness by using rotatory evaporator at 40ºC to yield (2.264 gm),as show in scheme (2-1). 18.104.22.168-Extraction method No.2: Fifty grams of powdered aerial parts of portulaca oleracea extracted by soxhlet with 500ml 0f 70% ethanol for 8 hr.