Then add two drops of iodine reagent to each of these wells and wait one minute, then add 0.5ml of amylase to each of the appropriate tubes. After two minutes, add a few drops from each tube, and place in the corresponding wall. After collecting the data use a color coding scheme to convert the qualitative data to quantitative data. The numerical data from each group was used to calculate the mean starch concentration and the standard deviation. The mean and the standard deviation values were calculated after entering all the data into
3.3 Colony PCR Single colonies were selected using sterile 10 μl pipette tip and inoculated with 0.2 PCR tube. Colony PCR was performed with each reactions consists of around 1 μl cell culture, 2.5 pmol of Grb14 forwa... ... middle of paper ... ...rm (1:1) was added to the linearized sample in a 1.5 ml microcentrifuged tube. The mixtures were centrifuged at 13000 rpm for 2 minutes at room temperature. The upper aqueous solution was transferred to another sterile 1.5 ml microcentrifuged tube. Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes.
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.
• The beaker was covered with the foil and stirred constantly on magnetic stirrer overnight (ideally 8 hrs) • After overnight extraction magnetic bead was removed and the extraction buffer was centrifuge... ... middle of paper ... ...ne (TMB) reagent • Stop solution 1. 2 N sulphuric acid • NUNC flat base ELISA wells PROCEDURE: • Prepare capture antibody solution of dilution 1:250 in coating buffer and add 100 µl in each well and incubate at 40C overnight. • Next morning aspirate and wash the wells 3 times with PBST • After washing add 200 µl blocking solution which is 10 % FBS in PBS in each well and incubate for 1 hour. • Aspirate and wash 3 times. • Add 100 µl standard and samples to the allotted wells.
2.4. Protein expression and western blotting HepG2 cells were seeded in a 12 well plate 24 hrs. prior to transfection. Cells were transfected with plasmids using lipofectamin 2000 (Invitrogen). After 48 hrs., total protein was extracted using lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris, pH 8.0).
100 uL of this solution was set aside in a tube labeled “HSS.” SDS-PAGE preparation. Eight microfuge tubes were taken and divided into two sets of four, labeled A and B. Each set had was labeled like the four we prepared earlier, “C”, “LSS”, “HSS”, “HSP.” 25 ul of the respective solution was pipetted into the two sets of tubes. Set A was given 25ul of 2X Laemli buffer A. Set B was given 25 ul of Laemli buffer B. Laemli buffer ( .125M Tris HCl, pH 6.8, 20% glycerol, 4% SDS, and 10% BME).
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.
Third, the remaining serum free medium and complete growth medium plates were analyzed. This assay was carried out by replacing the medium with 0.5 ml yellow MTT solution (Sigma) and incubated for two hours. Then, the MTT solution was replaced with 0.6 ml of MTT solvent (isopropanol) (BDH) and the plates were placed on a gyratory shaker for 15 minutes. Each well was split in triplicate in a 96 well plate in equal volumes. The absorbance was measured using plate reader at a wavelength of 570 nm.
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.
(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.