The flow through was discarded. 200 μl of A1 solution was added and centrifuged at 15 000 g for 2 min. The column was then placed in a new micro-centrifuge tube. 25 μl of DNA elution buffer was added directly to the column matrix and incubated at room temperature for 3 min. To elute the DNA, the columns were then transferred to 1.5 ml micro-centrifuge tubes and centrifuged at 15 000 g for 2 min.
After which the digestions were examined by gel electrophoresis. The samples were run on a 50 mL 0.9% (w/v) agarose gel in 1X TAE buffer at 100 V until the leading track dye traveled 2/3 the distance of the gel. The gel was then soaked in GelRed for 20 minutes and examined under UV light. To prepare the digestions 10 μL of each digestion was mixed with 2 μL of 6X track dye in a micro centrifuge tube. 12 μL of 1 kb DNA ladder and each digestion was run on the
The absorbance was read at 0 seconds, at 30 seconds, at 60 seconds, at 90 seconds and at 120 seconds. All the absorbances were remained 0 for the blank. After 120 seconds, the blank was then removed, and the appropriate amount of enzyme Tyrosinase (0.40 mL) was measured and added into the blank (cuvette #1) using the micropipette P-1000 according to the table 2. The final volume in the cuvette was 3mL. The cuvette contained the enzyme sample was wiped off with a KimWipe and was placed into the sample compartment of the machine.
Suspect one’s DNA was inserted into wells four and five; suspect two’s DNA was inserted into wells six and seven. The apparatus was then closed and turned on to run at 100 volts. Electrophoresis ran for 30 minutes; separating the DNA according to size in the gel. Then the tray with the gel was removed and a stain sheet was placed on the gel for 15 minutes. After 15 minutes that gel was rinsed with water and then set in water.
Second, 10.00 ml of the blue dye was poured into the 100.0 ml beaker and stirred for 2 – 3 seconds. The time taken by the solution to turn to colorless was measured with the aid of a stopwatch. The aim of this exercise was to determine the mixture that turned colorless in 15 minutes time. The data was recorded as shown in Table 2. Absorbance versus Time Measurements: The absorbance was set to 0 Abs while the spectrometer was set to ʎmax (from Part A).
The Solid sequencing platform, produced by Technologies/Applied Biosystems (ABI), performs sequencing by ligation method. Similar like the Roche 454 library preparation, genomic double strand DNA were sheared into small pieces and ligated with two types of adatptors P1 and P2 on two ends. One end with P1 adaptor binds onto the surface of the magnetic bead and emulsion PCR takes place to amplify single nucleotide fragment. Then the oil was washed out and four fluorescent labeled di-bases probes were added into the beads mixture. By matching the 1st and 2nd position of the template by di-base probes, fluorescence was detected and the extra tail with fluorescent probe is cleaved out.
Leah Romero 10/30/2017 Conclusion Lab 3 Chem 102L In lab 3, fundamentals of chromatography, the purpose was to examine how components of mixtures can be separated by taking advantage of different in physical properties. A huge process in this lab was paper chromatography, which was used to isolate food dyes that are found in different drink mixes. The different chromatograms of FD&C dyes were compared to identify which dyes are present in each of the mixes. Chromatograms where made for the known FD&C and for the three Kool-Aid samples. The retention factor for each dye was calculated.
The mixture was finally made upto 5 mL with distilled water and placed in hot water bath at 95ºC for 1 h. After cooling, 1 mL of distilled water and 5 mL of the mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was vortexed and after centrifugation at 4000 rpm for 10 minutes, the absorbance of the organic layer (upper layer) was measured in UV-Vis spectrophotometer (Shimatzu) at 532 nm against blank using distilled water. TBA when allowed to react with MDA aerobically formed a colored complex [MDA-(TBA) 2 complex] which was measured with spectrophotometer. MDA concentration (measured as TBARS) was calculated as
The cuvettes were retrieved from their respected conditions. 100 micro liters of solution C was added to cuvette 1b, 2b, 3b and 4busing a micropipette, the cuvette was covered with Para film in order to be mixed, then removed and was placed in the spectrophotometer. The absorbance was recorded immediately, then every thirty seconds for five minutes. Different volumes of solution C were added to cuvettes 1a-4a. 100 micro liters to 1a, 400 microliters to 2a, 200 micro liters to 3a and 500 micro liters to 4a.
The tube was placed back in incubation for 96 more hours to observe any more positives. 2.10 Catalase Test A trypticase soy agar plate was used and after incubation, four drops of 3% Hydrogen Peroxide was added to the plate to flow over the bacterial growth. A presence of bubbling was observed. 2.11 Starch Hydrolysis A starch agar plate was inoculated with a streak of the unknown bacteria and then incubated. On the second day of incubation, the plate was removed from the incubator and placed over a hot plate heating Iodine solids.