200 µl of lysis buffer (2 % Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl), 1mM EDTA, pH 8.0 and 0.2 g of glass beads were added to each Eppendorf tube. Then 200 µl of the solution phenol/chloroform/isoamyl alcohol (25:24:1) was added to the tubes under the fume hood and tubes were placed on rotator and left to mix for 3 min. 200 µl of TE buffer was added and spun for 5 min at maximum speed, the water phase was transferred to new tubes. 1 ml of cold 96 % ethanol was added, mixed and then spun for 5 min at maximum speed at 4°C. the supernatant was discarded and the pellet re-suspended in 400 µl of TE buffer (40 mM Tris-Base, 20 mM acetic acid, 1 mM EDTA, pH 8.0).
In the round-bottom flask (100 mL), we placed p-aminobenzoic acid (1.2 g) and ethanol (12 mL). We swirled the mixture until the solid dissolved completely. We used Pasteur pipet to add concentrated sulfuric acid (1.0 mL) to the flask. We added boiling stone and assembled the reflux. Then, we did reflux for 75 minutes.
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 aqueous extract was prepared by dissolving 1g of dry extract with 20 ml of sterilized distilled water, so the final concentration of extract would be 0.05 g/ml, from this solution other concentration were prepared (0.1-0.2) g/ml. the solutions were shaken for 30 min. The extract was centrifuged (30,000 rpm; 15 min) and the supernatant was Separated. To hydroalcoholic extract, 80 g of the powder was extracted with aqueous methanol (75%). The other two concentrations were prepared from soaking sixfold aqueous methanol (75%) with different amounts of powder.
We then added 10cm3 ethanoic anhydride to the salicylic acid and swirled the contents, this mixes together the two chemicals. We then added 5 drops of concentrated sulphuric acid to the flask and thoroughly swirled the mixture, this creates the solution that makes the aspirin. We then warmed the flask for 20 minutes in a 400cm3 beaker of hot water which was approximately 60°C, we made sure the flask did not go above 65°C because this could have caused the contents to evaporate. Part 2: Using a 25cm3 measuring cylinder we measured out 15cm3 of ethanol into a boiling tube and then prepared a beaker half filled with hot water at approx. 75°C, we got this temperature by filling the beaker with cold water and slowly adding boiling water from a kettle until we reached the right temperature.
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
3.3.4 Preparation of CaCl2 solution The molecular weight of CaCl2 was 111 g/mole. Weighed 11.1g CaCl2 and dissolved into 100ml distilled water. The final CaCl2 concentration would be 1M. CaCl2 solution was used to make bottom agar and initiate the infection cycle. 3.4 Selection of most sensitive strain to bacteriophages to make new stock culture Starting culture was prepared by inoculating 1ml (1×109 CFU/ml) stock Lactococcus lactis ssp.
5-aminotetrazole monohydrate: In a 250 ml round-bottom flask equipped with a condenser for refluxing (90 °C) and a magnetic stirring bar, 5.00 g (5.95 mmol) dicyandiamide (three times crystallized), 7.47 g (11.9 mmol) sodium azide and 11.00 g (17.8 mmol) boric acid and 100 ml of water is added and allowed to reflux for 24 hours, after the completion of the reaction, until the solution pH to about 2 to 3 as hydrochloric acid 37% is added (about 12 ml) Then the reaction mixture was cooled in a refrigerator for 18 hours and the white crystals formed. The mixture was filtered and washed three times with 10 ml of water and and dried in 60 °C for 5 hours and finally 45.8 g of product by it will be obtained. 5-Aminotetrazol monohydrate: Yield:,
The tube was placed back in incubation for 96 more hours to observe any more positives. 2.10 Catalase Test A trypticase soy agar plate was used and after incubation, four drops of 3% Hydrogen Peroxide was added to the plate to flow over the bacterial growth. A presence of bubbling was observed. 2.11 Starch Hydrolysis A starch agar plate was inoculated with a streak of the unknown bacteria and then incubated. On the second day of incubation, the plate was removed from the incubator and placed over a hot plate heating Iodine solids.
The natural agar consist of Yeast Extract, Tryptone , Lactose , Manniiol, Sodium Chloride , Dip otassium Hydrogen Phosphate ,Gelatin and Agar .Then , in a conical flask 14g of nutrient agar was mixed into 500 ml of distilled water and , The mixture was stirred and dissolved until most of the agar dissolve. By non-absorbent cotton wool plug the mouth of the flask was closed. By using the autoclave the agar was sterilized for 15 minutes.The cotton wool was removed. By the flame the mouth of the flask was heated before and after pouring the agar into the Petri dishes. And, the left hand the lid of the Petri dish was lift, just enough to enter the mouth of the flask and quickly was poured in agar (about 15 cm3).
The cuvettes were retrieved from their respected conditions. 100 micro liters of solution C was added to cuvette 1b, 2b, 3b and 4busing a micropipette, the cuvette was covered with Para film in order to be mixed, then removed and was placed in the spectrophotometer. The absorbance was recorded immediately, then every thirty seconds for five minutes. Different volumes of solution C were added to cuvettes 1a-4a. 100 micro liters to 1a, 400 microliters to 2a, 200 micro liters to 3a and 500 micro liters to 4a.
32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate. 50 μL of these dilution solutions were separated on the TLC plate coated with SNISG. The plate was developed with petroleum ether: ethyl acetate (4:1) and the movement of solvent was usually controlled at 1 cm from the upper edge. After completion, the plate was dried until no solvent smell remained. It was sprayed with an ethanol solution containing 10% sulfuric acid, and heated at an infra-red drier until obvious color came up, as shown in Fig.2 (B.ab).
Then, the pipet was rinsed with distilled water. The bulbs were then attached to the pipette; filling and dispensing water were practiced using both bulbs. Furthermore, the 250-mL beaker was weighed, and its mass was recorded. After that, the Erlenmeyer flask was filled with 100 mL of distilled water. The temperature was recorded.
Then carefully measure 25ml of methanol and 25ml of ethyl acetate using a measuring beaker 12. Pour each solvent into its respective labelled mortar 13. Stir each sample for 10 minutes using a stirring rod 14. Leave solutions in the sun for 12 hours, this is to allow for the active ingredients bond with the solvent and form a solution and to allow some of the Methanol and Ethyl acetate to evaporate. 15.