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).
The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water. The precipitate was dialyzed and used for enzyme purification using column chromatography. The dialyzed sample was loaded on DEAE-cellulose column, which was equilibrated previously with the buffer A (50 mM sodium phosphate buffer, pH 7.4). The column was washed with the five bed volumes of buffer A, and the enzyme was eluted with buffer A
2. FORMATION OF HYDRAZONE FROM ESTER Materials required: * The ester which was synthesized in the previous reaction. The total weight of ester obtained was 230mg. * Methanol – 20 ml * P-toluene sulfonylhydrazide (1.2 equivalent ) Procedure: * The ester was transferred in a round bottom flask and it was mixed with about 20ml of methanol and stirring was done on a magnetic stirrer till the ester dissolves in it completely. * In the above RB, a calculated amount of 1.2 equivalent amount of PTSH was added during continous stirring.
3) Centrifuged at 2500 rpm for 12 mins. Upper hexane layer (supernatant) was transferred carefully into another test tube. 4) Evaporated the hexane under a stream of grade 1 nitrogen gas and added 100 µl of methanol to the residue left and vortexed for 1 min. 5) Injected 100 µl of extract in HPLC vials and closed properly. Standard curves and calculations- Retinol was quantified from standard curves peak area for each vitamin.
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:,
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
Then, the test media is then incubated at 37 ° C, for 18-24 hours. Rinsing reusable instruments The samples were rinsed with 40 ml of pyrogen-free water using a glass beaker that is free from pyrogens. Endotoxin testing using STV A total of 0,2mL from the water obtained from the rinsing was placed in the STV containing LAL reagent and was shaken for 20 to 30 seconds. Then STV was placed in an incubator at 37 ° C for 60 ± 2 minutes. STV was then observed by reversing the reaction tube in one smooth motion.
Alginate sample (30 mg) was hydrolyzed in 10 mL HCl (0.3 M) at 100 ºC for 2 h. After cooling, the mixture was centrifuged (6000 rpm, 45 min), and the supernatant solution was separated and neutralized with 1 M NaOH and referred to as fraction A. The insoluble material was dissolved in 1 M NaOH and the pH was decreased to 2.85 by the addition of 1 M HCl. The suspension was recentrifuged and the supernatant was separated and referred to as fraction B. The insoluble fraction was dissolved by neutralization with 1 M NaOH and referred to as fraction C. The fractions A, B, and C are enriched in MG, MM, and GG blocks
The volumetric flask was then filled up to its 100 mL mark with deionized water. The buret was washed out with dionized water and then with the strong base NaOH before being filled up with NaOH. About 20 mL of the unknown weak acid was pipetted into a beaker. The starting volume of the NaOH in the buret was recorded before about 4 mL of the strong base was titrated into the weak acid solution. The final volume was recorded.
Ensure that solid is completely dissolved using a stirring rod. Next, a 10 mL beaker is filled with 3 mL of HCl and measure 10 mL of ionized water into a 140 mL beaker. Carefully turn on laboratory burner and start cleaning the Nichrome wire by dipping it into concentrated HCl acid. Hold the Nichrome wire on top of the flame and repeat the step until the wire doesn 't show any color. When the wire is clean, dip the wire again with some of the acid and dip it into the solution with the unknown compound in it.