Hypothesis: Increasing the temperature of Hydrogen Carbonate and Acetic acid should result in a faster rate of reaction (faster production of H2O, CO2, and C2H3NaO2). This is because when the temperature increases of the solution, the faster the particles move the more frequent the collisions between reactant particles. Safety precautions: When dealing with these solutions there isn’t very much to worry about, as the solutions are things that are worked with daily. These are water, vinegar, baking soda, and carbon dioxide. However when dealing with the hot plate gloves should be worn and the beakers needed to be carried with carefulness, as the beaker will be hot after being on the hot plate.
18.104.22.168 Gel extraction of amplified PCR products The amplified PCR product was eluted from the gel by using Quick Gel Extraction Kit (MN). The spliced gel piece was transferred to a micro centrifuge tube and added 3 volume of gel solubilization buffer to gel piece (approximately 100μl for 100mg). The sample was incubated for 10min at 50ºC water bath to dissolve the gel completely. The soluble solution was transferred to the spin column and centrifuged at 13000rpm for 2min followed by washing with 600μl washing buffer and centrifuged for 2min at 13000rpm. Then 30μl of TE buffer was added and centrifuged at 13000rpm for 2min, to get the DNA eluted from the column.
An aliquot (10 ml) was taken from the filtrate and titrated against standard sodium hydroxide using phenolphthalein as an indicator. The titrable acidity was expressed as percentage, citric acid and equivalent adopting the following formula. 22.214.171.124 Ascorbic acid (mg/100 ml) The volumetric method described by Ranganna (1991) was adopted. A known amount of the pulp was transferred to a 100 ml volumetric flask and volume made up with 3 per cent metaphosphoric acid solution. After 30 minutes, the suspension was filtered through whatman No.
Purification of glucoamylase The crude culture filtrate obtained after centrifugation was concentrated 5 times using rotary vacuum evaporator (EYELA Rotary Evaporator N 1000 Japan) followed by precipitation of enzyme at 40C using chilled acetone16. Precipitated protein was collected by centrifugation at 5,000 rpm for 15 mins at 40C. The precipitate was resuspended in 10 ml 0.1 M acetate buffer (pH 4.8) and enzyme activity was measured. DEAE-Cellulose chromatography 10 ml of the resuspended precipitate was applied to the previously equilibrated DEAE-Cellulose column 17. The enzyme was eluted with 0.1 M acetate buffer (pH 4.8) contaning a linear gradient of (0-0.1M) Nacl at a flow rate of 20 ml/h.
3.9.3 Ion Exchange chromatography As maximum activity was observed in the 50-80% fraction obtained from sequential ammonium sulfate precipitation, this fraction was subjected to ion-exchange chromatography on a DEAE-Cellulose column equilibrated with 0.15 M PBS. The resin was treated and column was packed as per the manufacturer’s instructions. • 5gm dry resin was suspended in 25 ml of distilled water and left at room temperature for 30-45 minutes to settle down the matrix. The settled volume of the resin was measured and this was termed as Column volume (CV) and used as a measure for washing. • Further the filter resin was suspended in 2 CV of 0.15 M NaOH containing 0.5 M NaCl for 10 minutes, and washing was continued for 5 CV of 0.15 M
From the standard curve, there is one equation which is y=1.0685× to calculate the concentration of nitrate. Another way is applying the formula that is concentration (mg/ l) = sample absorbance x F factor. The F factor is calculated by using 1.0 mg/l / (Es-Eb). From two ways to determine the calculation, the equation from the standard curve is more accurate than the formula of concentration. This is because all the reading is recorded step by step and the standard curve have done after experiment.
Each isolates was inoculated into 50ml cellulase enzyme production broth medium with inoculum size of 2 x 106 spore concentration. 3.1.10 Preparation of crude enzyme After a desired period of incubation, the cellulase enzyme production broth was poured into a sterile Falcon tube and centrifuged at 10 000 rpm at temperature of 5oC to separate the cell and the broth. The supernatant was kept as a crudes enzyme sources for enzyme assay. 3.1.11 Preparation of sodium citrate buffer 3.2 Analysis techniques 3.2.1 Reducing Sugar Assay
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
Briefly, 1.5 ml of DPPH solution (0.1 mM, in 95 % ethanol) was incubated with different concentrations of unirradiated and irradiated CMCS solutions. The reaction mixture was shaken well and incubated for 15 min at room temperature and the absorbance of the resulting solution was read at 517 nm against a blank (control). Ascorbic acid was used for comparison as antioxidant materials. The radical scavenging effect was measured as a decrease in the absorbance of DPPH and can be calculated using the following