Preparation Of Lb Broth And LB Agar Preparation

One g of material was ground and extracted with 0.2 N HCl by continuous shaking. To 0.2 ml of the filtrate, distilled water to make volume 1.4 ml was added. After that1ml of ferric ammonium sulphate solution (21.6 mg in 100 ml water) was added, mixed and placed in a boiling water bath for 20 min. The contents were cooled and 5 ml of isoamyl alcohol was added and mixed. To this, 0.1 ml ammonia solution was added, shaken thoroughly and centrifuged at 3000 rpm for 10 min.

Characterization of Aspirin. In the first section, the Synthesis of Aspirin, salicylic acid was weight to be 3.029 grams using mass by difference since it was weighed on a 150 milliliter beaker. 9.23 milliliters of the acetic anhydride and 14 drops of 85 percent phosphoric acid were added to this beaker. A Bunsen burner provided by the laboratory was then used to boil the just mixed combination by producing a flame underneath the positioned beaker on top, and then allowed to cool for several minutes after the Bunsen burner flame was terminated. Two quantities of distilled water were then added to this mixture to make it cool even further, which were 41 drops and 30 milliliters.

• Add 100 µl standard and samples to the allotted wells. • Incubate overnight at 40C. • Aspirate and wash 5 times with PBST • Add 100 µl of secondary plus detection to each well and incubate at room temperature for 2 hours. • Aspirate and wash 7 times with PBST. • Add 100 µl of substrate solution to each well and allow it to develop for 30 minutes in dark.

Then transferred each beet to a separate test tube containing deionized water. After 20 minutes in these diffusion solutions, we took the beets out with a dissecting needle and discard it. We then stirred each solution in the test tube with a stirring rod, and transferred it to a cuvette. A spectrophotometer was then calibrated, and used to measure the absorbance of each exposure solution, and diffusion solution. Membrane Damage For the lab experiment for Membrane Damage, we tested the extract pigment and diluted it.

(SDS denatures the proteins, BPB is used as a tracking dye while Beta Mercaptoethanol breaks sulfide bonds.)  Heat the samples at 80 degrees Celsius for 5 minutes in a water bath. This helps in protein denaturation.  Put the cassette into the buffer dam and fill it with the running buffer. For SDS-PAGE, running buffer is prepared by taking 40 ml Tris-Glycine and adding 4 ml of 10% SDS to it.

A tripod and Bunsen burner is set up and the beaker is heated with the blue Bunsen burner flame until it nearly boils. Then a spatula is needed to add parts of the copper(ii) to the beaker, continuously stirring for about thirty seconds and adding the next portion, and stirring and so on until all of the copper oxide has been added. The beaker is then heated further for a couple of minutes, to ensure the copper oxide has completely reacted with the sulphuric acid. The next stage of the experiment requires a filter funnel to be placed in the neck of the conical flask. Filter paper is to be folded into the funnel.

Now heat to 80-85°C over 10 minutes and hold there for at least two minutes but not more than five. Cool in water to room temperature. Transfer the solution to a 100 mL beaker, keep some (~2-3 mL) in the Conical flask. Stir at medium speed on a magnetic stirrer. Now add 30% hydrochloric acid drop by drop till pH of the solution become 7 and filter it.

The measurement was .995g. The salicylic acid was transferred into a 125 ml Erlenmeyer flask and the acetic was given by the lab instructor. Three drops of 85% phosphoric acid. While swirling and mixing more was added and then clamp onto the sing stand in the boiling water for 8 minutes. 1 mL drops of water was added in the flask.

After the calibration process, few drops of each solution were transferred from each test tube without removing the test tubes from their temperatures. These drops were added into the first row of wells on the spot plate, then add two drops of iodine to the wells on the spot plate and wait 1 minute. Use a color- coding scheme to convert the data to qualitative data into quantitative data this will serve as the control. Add 0.5 ml of amylase to the appropriate test tube containing starch and wait two minutes. Then add two drops of the starch-amylase mixture from each tube to the third row of

0.5ml of extract along with 0.1 ml of (0.5N) Folin-Ciocalteu’s reagent was incubated at room temperature for 15 min. After incubation, 2.5 ml of saturated sodium carbonate solution was added and further incubated for 30 min at room temperature. The absorbance was measured at 760 nm. Gallic acid was used as a positive control. Total phenol content was expressed in terms of gallic acid equivalent (mg/g of extracted compounds).The assay was carried out in triplicates and expressed as mean±SD.