Cell Lab
Materials and Methods and Results for lab 4.
Materials and Methods
Homogenization
Spinach leaves were used. The large veins were removed and the sample was weighed. Four grams were used to start. The leaves were chopped finely and added to an ice-cold mortar with 15 ml of homogenization buffer( .33M sorbitol, .05M HEPES, 4mM MgCl2, 2mM EDTA, pH 6.5).
The leaves were grinded to a paste and filtered through two layers of cheesecloth. The filtrate was collected, and each lab group was given a 1.5 ml aliquot of the filtrate. We took a 100uL of this solution and put into a microfuge tube labeled “C”.
Centrifugation
The microfuge tube was set aside on ice while the remaining filtrate was centrifuged at 200g’s.
The supernatant was removed and 100ul were set aside in a tube labeled “LSS” (for low speed supernatant) on ice. The remaining supernatant was centrifuged at 1000g’s for 7 minutes. 100 uL were taken from supernatant, and placed in a microfuge tube on ice labeled “HSS.” The pellet was resuspended in 250 ul of homogenization buffer. 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 of the two buffers was made without BME, which breaks disulfide bonds. The buffer without BME will be determined by SDS-page analysis.
The samples were boiled for 5 min, and stored at 20 degrees C for one week.
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.
The incubated samples were placed in the model 340 spectrophotmeter and read at 562 nm. The readings were taken, and the protein amounts were calculated using a standard curve( y = .
In order to make sure that no sediment was transferred into the clear supernatant, brieflycentrifuge the supernatant. Another way to improve this procedure would be to collect 0.50 mL of supernatant using a 0.50 mL pipette; this would limit the amount of inaccurate readings of the volume. Ultimately, causing the densities to be more accurate. Additionally, to improve this procedure, two weeks could be allowed for the fermentation reaction to sit instead of one week. This is because one week is the minimum amount of time required to let a fermentation reaction sit. Therefore, the longer the reaction sits, the more likely it will be to obtain a high percent yield of
Every day of observation, the liquid treatments were applied to the plants.. A spray bottle was used to administer gibberellic acid, Cycocel, or water to the plants. One bottle contained only water, another contained 100 parts per million of gibberellic acid, and the other contained a 1:100 dilution of Cycocel also known as B-9 solution. Two full squeezes were dispensed over each of the four sections of a occupied quadrant. The nozzles were sprayed into a small plastic tube to avoid cross-contamination with adjacent quadrants. The plants were kept under fluorescent light on a timed cycle where they were on for 16 hours of the day and off for the other
The purpose of this lab was to extract chlorophyll and carotenoid pigments from fresh spinach leaves and separate and analyze these pigments using column chromatography and thin layer chromatography. Acetone was used as a polar solvent to dissolve the more polar pigments first (Xanthophylls, chlorophylls), while hexane was used as a nonpolar solvent to dissolve the more nonpolar pigments such as the carotenes. In addition to being used as the polar solvent, acetone was used to remove the spinach components that were not pigments such as cellulose which is insoluble. The column chromatography worked by eluting the nonpolar carotene pigments first because the alumina is polar and doesn’t absorb the nonpolar carotene. The polar components such
Aluminum foil was not used during the experiment to wrap around the Hickman still. The temperature of the hot plate continuously increased because the thermometer temperature increased very slowly. The stillhead started to collect condensate around 50°C which is much lower than the 80-90°C that the thermometer was supposed to read. A bent pipette was used to collect around 1.5mL of distillate that was put into a .5-dram vial. This vial was named “Fraction 1.” The hot plate temperature was increased a little more to increase the thermometer temperature in the Hickman still. .6mL of distillate was collected, using the same bent pipette, and put into another .5-dram vial named “Fraction 2.” The hot plate was turned off, and everything was left to cool for a little bit. After it cooled, around 1.2 mL of clear liquid was left in the original vial and transferred to another vial named “Fraction 3.” The syringe for gas chromatography was flushed with acetone and then with Fraction 1 before any reading was done. Because the syringe only collects .2µL of Fraction 1, the liquid wasn’t visible to the naked eye. The needle had to be twisted and rotated gently to insert into the injection port. When the LabQuest collected to data, there was one tall peak and a few little ones. The Percent Area for peak 1 was -197.78 and peak 2 was 297.78, and the Retention time for peak 1 was .700
Step 5: Using the tube at room temperature (23C) repeat step 3 for this tube and
When reproducing this experiment several changes should be made. One change could be applying the salt to full grown plants to more accurately simulate a real life
Then add growth media until the sample is submerged beneath the 2-3 ml of liquid, cap the tube and invert several times to mix thoroughly, incubate the tube while shaking vigorously in a shaking incubator at 250 rpm for a couple hours, then allow the sample to sit.
Two spinach leaves were extracted and grinded using a pestel. The spinach leaves were then mixed with methylene chloride and added to separatory funnel along with water. The organic (green) layer of spinach leaves was then allowed to drain and concentrated in the hood. A melting point capillary was then used to insert some organic solution onto the TLC plates. These TLC plates were then placed in solutions of different ration of acetone and hexane. In this case, the stationary phase was the silica gel plate and the mobile phase was the solvent. According to our observations 4 acetone: 6 hexane and 6 acetone and 40: hexane solutions were able to show the color bands for most spinach pigments. However, the ratio of 4:6 didn’t show the bands for xanthophylls and carotene while 6:4 ratio did show the band for yellow and light yellow which are characteristic of xanthophyll and carotene. We also calculated the Rf factor by measuring how far the substance travelled (ds) and how far the solvent travelled (dx) (Rf=ds/dx). By calculating the Rf values in 6:4 ratio, we can conclude that Xanthophyll (Rf=.84), carotene (Rf=.8), Pheophytin (Rf=.62) were more polar than chlorophyll A (Rf=.89) and Chlorophyll B (Rf=.87) because they had lower calculated Rf
In this investigation, there were some limited factors. The experiment of each substance was done by different groups, which could have affected the experiment on factors such as how well the bacteria were spread across the agar plate. Also, the procedure stated to evenly spread the filter disks; the distance was not measured during the experiment. This could have had impact on the results, as some disks may have been closer to each
For the first dialysis tube, which contained the 10 mL of 10% sucrose, it was hypothesized that water will move from the outside of the dialysis tube to the inside due to the distilled water in the beaker being of a higher concentration than within the dialysis tube. Once the experiment was completed it was shown that the weight of the dialysis tube had gone up almost a full mL. Based upon this information it can then be determined that the solution within the dialysis tube was hypotonic and the distilled water in the beaker can be seen as hypertonic.
In this experiment, in the first part, the best concentration of enzyme was determined by recording the absorption over time. In the second part, the best concentration was selected from the previous experiment which was C and the optimum pH was determined.
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
Note: The revised version of the method (9.1.) differs in one major point from reference 9.4.: Preparation of samples and standards for analysis. It is now ...
The Cell, the fundamental structural unit of all living organisms. Some cells are complete organisms, such as the unicellular bacteria and protozoa, others, such as nerve, liver, and muscle cells, are specialized components of multicellular organisms. In another words, without cells we wouldn’t be able to live or function correctly. There are Animal Cells and Plant Cells. In Biology class the other day we studied the Animal Cell. We were split into groups of our own and we each picked a different animal cell slide to observe. My group chose the slide,'; Smeared Frog Blood ';.
After 30 minutes, another 5 ml of acetic acid was added, followed by 1.5 g of NaClO2the following 30 minutes. These steps were repeated until a total of 6 g of NaClO2 was added. The mixture was heated for a further 30 minutes after the final sodium chlorite addition. The suspension was then cooled in an ice bath before being filtered using sintered glass crucible and rinsed with cold distilled water. A final wash was carried out using acetone. The crucible with holocellulose was air dried in an air-conditioned room until constant weight was achieved for further alpha-cellulose analysis. For hemicellulose determination, the oven-dry weight of cellulose was used for