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
Punch disks of filter paper using a paper punch 2. Add the various amounts of medication solutions as shown in table 1.2 below to each filter disk using a micropipette 3. Place filter disks in a sealed container and leave for 24 hours Suspension of bacteria 1. Unseal the petri dishes 2. Add 1000 microliter of 0.8% of saline solution to the centre of the first petri dish using a micropipette 3.
Then get the Acetic acid and pour exactly 100mL of it into the graduated cylinder (This should almost fill it up). When that is completed repeat steps 1 and 2 four more times, which would mean five graduated cylinders of 100mL of Acetic acid. Part 3: Carrying out the reaction Wash the 250mL-beaker and dry it with paper towels. Add one of the 4.2 grams of catalyst (Sodium Hydrogen Carbonate) that has been obtained in step one into the beaker. Making sure that the beaker is dry before hand.
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
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 dried mushroom samples were further heated at 105 ºC overnight until constant weight obtained for moisture content determination. The samples were incinerated at 550 ºC for 24 h and reweighed to determine ash content. MicroKjeldahl method was employed for the crude protein content (N × 4.38). The crude fat was determined by extracting a known weight of sample in Soxhlet apparatus using petroleum ether as a solvent. Total carbohydrates calculated by the difference.
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.
Yeast Growth in YPD Agar: To prepare YPD agar, mentioned in Table 1 nutrient ingredients in given concentration were weighed and added to 200 ml of distilled water. The mixture was autoclaved (SMS ASL80 MSV) for 1.5 hours at 121°C. On sterile plates, 25-30 ml of the mixture was poured and left to cool down. The yeast cells were then streaked on the agar plates and the cultures were grown in a stationary incubator (S1-600R, Lab Companion) for 72 hours at 30°C. Yeast Growth in YPD: To prepare YPD liquid medium, glucose, peptone and yeast extract were weighed and added to 200 ml of distilled water.
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