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.
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.
The completely dried and activated plates are kept in a dry place for use. The crude extract applied in a row or bands of spots as a concentrated solution by using the capillary tube at least five times on each plate with concerning the drying of each spot before the another application.The solvent system (S5) ( rutin and quercetin) , and S7 (genistein), put in a glass tank (22.5 x22 x7)cm, covered with glass lid and allowed to stand for 45 min.in order to saturate the tank before use. By the aid of Liebermann burchard reagent and UV at 254 nm the bands can detected. The target bands after detection were scratched off the developing plate , collected in dry, clean beaker and mixed with mobile solvent of chloroform- methanol (60:40), then heated with continuous stirring and filtered. After the evaporation of the mobile solvent
The mixture was finally made upto 5 mL with distilled water and placed in hot water bath at 95ºC for 1 h. After cooling, 1 mL of distilled water and 5 mL of the mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was vortexed and after centrifugation at 4000 rpm for 10 minutes, the absorbance of the organic layer (upper layer) was measured in UV-Vis spectrophotometer (Shimatzu) at 532 nm against blank using distilled water. TBA when allowed to react with MDA aerobically formed a colored complex [MDA-(TBA) 2 complex] which was measured with spectrophotometer. MDA concentration (measured as TBARS) was calculated as
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.
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
Measure 50 ml of distilled water using the measuring cylinder. Pour this amount of water into the beaker. Drop exactly one drop of food colouring into the water in the beaker and wait until the whole liquid changes colour. Pour the liquid in the beaker inside the wine glass and leave it on the side. Setup the oscilloscope and connect the microphone to it.
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.
After 10 minutes, the solution was deleaded by adding potassium oxalate crystals in excess and the volume was made up to a known amount with distilled water and filtered through whatman No. 1 filter paper. The filtrate was taken in a burette and titrated against boiling Fehling’s mixture (5 ml of Fehling’s solution A + Fehling’s solution 5 ml of B) till the blue colour faded. Then one ml of methylene blue indicator (1 per cent) was added and the titration was continued till the contents attained a brick red colour and titre value was noted. The percentage of reducing sugar was calculated according to the following
Subsequently, monochloroacetic acid (60 g) was added, in five equal portions, at 1 min intervals. Heat was then applied to bring the reaction mixture to a temperature of 60 oC and stirring at this temperature was continued for 3 h. Afterward, the reaction mixture was filtered and the filtered solid product (CMCS) was thoroughly rinsed with methanol. The resultant CMCS was dried in an oven at 60 oC. The Mw of the produced