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.
Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes. Again, the aqueous solution was transferred to a new 1.5 ml microcentrifuged tube and 1/10 volume of 3M Sodium Acetate Solution was added. Then, 2.5 volumes of cold absoluted ethanol were added to precipitate the DNA. The mixture was incubated in -20 °C for overnight or -80 °C for 1-2 hour. After incubation, the mixture was then spinning at 13000 rpm for 30 minutes at 4 °C.
This was to see the difference between the initial temperature and the final temperature. First Test In a 250ml beaker place 100mls of water, measure the temperature of the water and record this initial temperature onto a table. Set the timer and add one teaspoon of Ammonium Nitrate to the water, stir this continuously until the Ammonium Nitrate has dissolved. After 1 minute measure the temperature and record it, do this for a further 2 minutes (3 minutes in total). Repeat this process for a total of 10 teaspoons.
(1999). Freeze-dried avocado mesocarp (0.05 to 0.10 g) was mixed with 10 mL 80% (v/v) ethanol and homogenized for 1 minute. Thereafter, the mixture was incubated in an 80oC water bath for 60 min to extract the soluble sugars. Subsequently the mixture was kept at 4 ºC overnight. After centrifugation at 12000 g for 15 min The mass of each fruit was weighed before cold storage using a weighing scale.
For SDS-PAGE, running buffer is prepared by taking 40 ml Tris-Glycine and adding 4 ml of 10% SDS to it. Its volume is raised up to 400 ml by adding distilled water. Load these samples into wells created in the gel. Run the electrophoresis unit at 5 mA and 20 V for entry of sample into lower gel. Once the samples enter the lower gel, alter the current to 10 mA and set the voltage at 90-100 V. Leave this setup undisturbed for around 3 hours.
28-30 C) for 24 hours or until the mixture reaches the chloroform number of 3 and % quick swelling in toluene of 90-95%. Methods of measurements of chloroform number and % quick swelling are given in Sections 4.8.7 and 4.8.8, respectively. Table 4.4 Formulation for preparation of prevulcanised
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.
Firstly, The Strata-X-A SPE cartridges prepared using 3 mL of methanol and equilibrated followed with 3mL of ultrapure water. Next, buckwheat honey samples of 30 g were thoroughly mixed with 120 mL of distilled water, and subsequently the solution was adjusted to pH 7.0 with with 5% ammonium (v/v).The buckwheat honey samples were centrifuged with 8000 × g for 10 min to dislodge the solid particles. The supernatants were was passed through the previously conditioned Strata-X-A SPE cartridges. After the samples was loaded, the columns were washed 4 mL of distilled water for Strata-X-A SPE cartridges to remove compounds of buckwheat honey that were not absorbed on the sorbents . Afterwards, the phenolic compounds were eluted with 5 mL of formic acid:methanol (1:9, v/v).
Protein quantification Five tubes labeled “Blank”, “C”, “LSS”, “HSS”, “HSP” were obtained. The blank contained 100 ul of distilled water. The others had 90 ul of distilled water and 10 ul of the cellular fractions obtained by centrifugation. #ml of BCA were added. The samples were placed in a water bath and incubated at 37 degrees Celsius for 30 min.
Next, 0.55mL of 0.1M IPTG was added to each culture and placed in the shaking incubator at 37°C for one hour to prompt the expression of peptides. The induced cultures were centrifuged at 4°C for 20minutes at 4000rpm, and the pellets were stored at -20°C until use. Lysis/Binding buffer was made with 5µL of 5M imidazole and 5mL of Common buffer (500mM NaCl and 50mM Tris-HCL at pH7.5) and left on ice. 1mL of this buffer was added to thawed 6xHis-USP7 pellets. The mixture was vortexed, placed on ice, and lysed with an enclosed sonicator for four 20 second pulses at 15 second intervals, at 30% intensity.