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
Animals were also weight immediately prior to sacrifice (fasted body weight). Animals were sacrificed under anesthesia with diethyl ether, and then blood samples were immediately collected in clean and dried Wiesserman tubes from the portal vein. First part of blood was collected in tubes containing potassium oxalate and sodium fluoride for the estimation of plasma glucose by O-toluidine method of Sasaki et al. (1972). Second part of blood was left to coagulate then centrifuged at 3000 rpm for 15 minutes to obtain serum to estimate some biochemical parameters.
Fertilization is carried out by adding one ml of sperm solution with approx. 100x106 sperms to the mentioned ovules (1 million) which means a ratio of 100 sperms to each ovule. The sperm solution has 1 ml of concentrate of sperms obtained directly from the gonad of the sea urchins and diluted in a volume of 200 ml. The success of fertilization as well as embryonic development is determined through microscopic examination. After 30 minutes the fertilization, fertilized eggs (embryos) are poured out whereas one-half of the water is changed with fresh, 0.5 um-filtered and UV irradiated seawater at 17-18
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.
Experimental protocol: Animals were acclimatized for a period of 1 week prior to the experiment. Later they were divided in to five groups containing six animals each. For induction of lung carninogen BALB/c mice were given Urethane in all the group except normal control, twice a week for a period of 4 weeks by intraperitonial injection. Group I serves as normal control receive normal saline. Group II serves as Disease control receive Urethane at a dose
1.3.3 Methods of the measurement of DNase activity In 1950, the first method of the measurement of DNase I activity was described by Kunitz196. He isolated and precipitated DNase from fresh beef pancreas and isolated thymus nucleic acid. He found that the cleavage of DNA by crystalline DNase is accompanied by increase of absorption (at 260 nm) of UV light. This spectrophotometric method of measurement of the rate of the increase in the light absorption was then used for estimating of DNase activity. It lasted 43 years until in 1993 Nadano et al.
Placed the four test tubes into foam microtube holder and placed in water bath to incubate at 37 degrees Celsius for 45 minutes. Ms. Lovrien added 5 L of 10x loading solution to tubes labeled 1-4. Placed samples in the refrigerator overnight. After returning to class the next day, obtained a gel tray that contained 0.8% agarose with TAE buffer. Removed that comb and tape.
Single cell DNA Fingerprinting- Dr Ian Findlay and his colleagues first reported the flourishing development of a DNA fingerprint from a single cell in 1997. Single-cell DNA profiling is mainly helpful in rape cases, as DNA in sperm cells is extremely conserved due to it being so compressed in the protein head. There is also likely for the method in use in documents. 9. Mitochondrial DNA-Mitochondrial organelle is concerned with the making of cell energy.
Worms A, B and C were then placed into separate containers containing the caffeine treatment solution. After allowing the worms to be treated for fifteen minutes, they were briefly rinsed and placed in the viewing chamber to measure their pulsation rate. This was done three times for each worm to calculate a mean rate for first level of treatment, 3.0 mM of caffeine. The same procedure was repeated for part II of the lab, however; a different set of three worms were treated with nicotine and all the means were collected to calculate the standard deviation of each
The animals were fasted for 24 h before the experiment with free access to water and were treated orally with two equal doses of either indomethacin (10 mg Kg-1 b.w.) or the test compounds (50 mg Kg-1 b.w.) at 0 and 12 h intervals, except the control group, which received 0.5% w/v CMC. After the drug treatment, the rats were fed a normal diet for 17 h and then sacrificed. The stomach was removed and opened along the greater curvature, washed with distilled water and cleaned gently by dipping in normal saline.
After adding 20 microliters of proteinase K to the 1.5 ml tube, the sample was vortexed for 30 seconds to disrupt the pellet. The sample was then incubated at 56°C to lyse the tissue. The sample was checked every fifteen minutes and vortexed between each checking for an hour and a half until the tissue was completely lysed. The tissue sample was then again vortexed. Next 200 microliters of buffer AL was added and
Once the gel hardened, .5X TBE (44.5 mM Tris base, 44.5 mM boric acid, and 1.0 mM EDTA) was added just until the gel was covered with the TBE buffer. Each sample was loaded into the gel as well as 10 μL of DNA size markers (1kb ladder, New England Biolabs) into a separate lane. The gel was allowed to harden at room temperature and then electrophoresed at 100 volts for 75 minutes. Using a UV imager, a photo was taken of the resulting traveled DNA fragments in the gel. Results Table 1.
32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate. 50 μL of these dilution solutions were separated on the TLC plate coated with SNISG. The plate was developed with petroleum ether: ethyl acetate (4:1) and the movement of solvent was usually controlled at 1 cm from the upper edge. After completion, the plate was dried until no solvent smell remained. It was sprayed with an ethanol solution containing 10% sulfuric acid, and heated at an infra-red drier until obvious color came up, as shown in Fig.2 (B.ab).
The DNA copies are doubled with each repetition of the three steps. The PCR products are usually analysed using Agarose gel electrophoresis methods, which separates the DNA fragments based on size. A study carried out at the University of Sydney under the National Myrtle Rust Transition to Management (T2M) Program used polymorphic satellite markers technique to understand the genetic variability of Myrtle rust isolates. The amplification of the satellite markers was done using PCR with specific controls. Reactions were performed in a 96-well DNA thermo cycler (Eppendorf Mastercycler, Germany) using the following reaction mixture: • 2.0 μl of genomic DNA (10 ng/μl) • 1.5 μl of 10x PCR buffer (NH4 Reaction buffer, Bioline) • 1.5 μl of dNTPs (0.2 mM) • 0.9 μl of 50 mM MgCl2 (Bioline) • 0.9 μl of each forward and reverse primer (2 mM) • 0.15 μl (5 u/μl) of Taq DNA (Bioline, Australia) • 7.15 μl of