The pK184hlyA and pBluescript plasmids were cut using EcoR1 and Pst1 restriction enzymes that cut at specific restriction sites. Gel electrophoresis was carried out to check if the digestion procedure was done successfully.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
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Haemolytic colonies were classified by a white ring around the patched colony, indicating that haemolysis of the blood agar occurred. Conversely, non-haemolytic colonies were classified by a lack of a white ring, which indicated that no haemolysis took place.
While the previous experiment identified colonies containing recombinant DNA, the patching experiment distinguished which colonies contained the hlyA fragment and which ones did not. Colonies that could cause haemolysis of the blood agar plate indicated that recombinant DNA taken up contained the hlyA fragment ligated with pBluescript, which is the desired subcloning product. The hlyA fragment contains the hlyA gene which encodes for a haemolytic protein that causes the red blood cells in the blood agar to lyse. Therefore, non-haemolytic colonies were transformed with pBluescript plasmid ligated with the pK184 fragment and were not able to cause haemolysis as no hlyA gene was present. In theory, this experiment allowed for the aim to be achieved as it identified colonies with the desired product. Inoculating certain colonies in broth culture allowed for gel electrophoresis to be carried out and confirm if the aim of the experiment has been
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Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
In this experiment, we determined the isotonic and hemolytic molar concentrations of non-penetrating moles for sheep red blood cells and measured the absorbance levels from each concentration. The results concluded that as the concentration increased the absorbance reading increased as well. A higher absorbance signifies higher amounts of intact RBCs. The isotonic molar concentration for NaCl and glucose is 0.3 M. The hemolysis molar concentration for NaCl and glucose is 0.05 M. Adding red blood cells to an isotonic solution, there will be no isotonic pressure and no net movement. The isotonic solution leaves the red blood cells intact. RBC contain hemoglobin which absorbs light, hemoglobin falls to the bottom of the tube and no light is absorbed. Determining the isotonic concentration of NaCl and glucose by finding the lowest molar concentration. In contrast to isotonic molar concentration, hemolysis can be determined by finding the
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
a) Urinalysis with significantly increased amounts of blood (via dipstick and sediment), protein, and leukocytes as well as slightly increased bilirubin and slightly decreased pH;
The gels were run at 90-100 volts for 1-1.5 hours. Upon completion of the experiment, we were able to examine the DNA. First, the electrophorese. revealed that three of the fourteen samples were homozygous while the other eleven were
Implementation and Data Presentation The counting chamber of the Haemocytometer is 0.1 mm deep. The central squared area is divided into 25 main squares, each of which is subdivided into 16 smaller squares. The volume of suspension above the smallest squares is 0.00025 mm3. So the number of cells in the culture suspension is given by.
However, it is only a certain kind of white blood cell that comes out in the non specific response, and it is called a phagocyte - meaning 'cells that eat'. In a process known as phagocytosis, phagocytes can envelope any kind of pathogen within the body and, whilst the pathogen is inside the phagocyte, it can destroy it.
Immediately after wounding, the first phase of hemostatsis sets in motion with vascular constriction which restricts the blood flow in the blood vessels followed by the platelets plug formation which creates a temporary blockage of blood flow and then coagulation takes place with fibrin clot formation. The clot and surrounding tissue release pro-inflammatory growth factors and cytokines such as transforming growth factor (TGF)-13, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF).
In figure 1.2, a major correlation between absorbance and percent hemolysis cannot be seen because the values fluctuated tremendously. On the contrary, it follows a pattern whereby a large value of absorbance gives a large value of percent hemolysis. In tubes 1 through 6 and the control hemolysis occurred, but in tubes 7 through 11 the red blood cells remained the same. The calculated osmolarity increased from 31.034 mOsm/kg to 341.352 mOsm/kg, thus displaying a negative slope between osmolarity and percent hemolysis. In other words, the NaCl concentration on the outside of the erythrocytes was larger than the inside, thus causing osmolarity to increase. Therefore, the green light from the colorimeter did not absorbed as much erythrocytes because water was unable to permeate the cells. In solutions 10 and 11, absorbance nor hemolysis did not take place because of the 0 values which means that the red light completely transmitted through these red blood
In the first experiment we measured the amount of maximum absorbance of the RBC’s that were lysed and due to blood being red we expect to see a absorbance peak for the hemoglobin at around 540nm, but it could range between 500nm to the 550’s. We hypothesized that the solutions that contained minute NaCl (0.09,0.18,0.27,0.36,0.45,0.54,0.63, and 0.72% NaCl solutions) should have hemolysis readily occuring, and that it should also only occur slightly in solutions with higher concentrations of NaCl (like .81 and .9% NaCl solutions) because of the presence of the hypotonic solutions. We also hypothesized that hemolysis of cells would occur more quickly in the 0.9% NaCl plus one drop of saturated soap solution and that in the 0.9 NaCl (standard) solution there would be no hemolysis because it aids in the production of a isotonic environment. We also hypothesized that hemolysis of the 0.3 M ethylene glycol solution would be quick and 0.3 M glycerol and 0.3M glucose solutions would occur much more slowly based on their structure and hydroxyl
Restriction enzyme will cleave a piece of DNA into a series of fragments. The number and sizes of the fragments depend on the number and location of restriction sites for that enzyme in the given DNA. A specific combination of bases will occur at random only once every few hundred bases, while a specific sequence of 6 will occur randomly only once every few thousand
The Use of Recombinant DNA Technology Recombinant DNA technology is the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.(1) This is the process of using recombinant DNA technology to enable the rapid production of human protein from a single gene of insulin. Firstly the single gene required must be isolated. This can be done three ways: Either by working backwards from the protein- Finding the amino acid sequence for the protein needed, the order of bases can be established using known genetic code. New DNA can be made from this sequence of bases resulting in artificial gene made from complementary DNA.
I. Plasmids are important tools in molecular biology. Plasmids are small circular DNA that has the ability to enter and replicate in bacterial cells and can be used as vectors to introduce foreign genes into bacteria for cloning and sequencing. Any gene must be inserted into an appropriate location of a plasmid to be expressed. The importance of a plasmid is in the step of cloning and sequencing when the construction of a recombinant DNA molecule occurs. The target gene fragment is ligated to a plasmid, and becomes recombinant DNA. Then the plasmid can replicate autonomously in an appropriate host organism.
Recombinant DNA technology has opened the door for humans to isolate and purify virtually any known genomic sequence. The human genome is known to contain approximately 6x109 base pairs over a span of 23 pairs of chromosomes. Getting pure DNA samples from large genomes like ours is now made far easier thanks to recombinant DNA technology. In addition, functional regions can be investigated and studied in predetermined manners, giving us vital insight to the biochemical, molecular, and genetic properties of our DNA (Lodish et al., 2000). Recombinant DNA itself is any DNA molecule formed by joining DNA fragments from different sources. The most frequent manner that recombinant DNA is produced is by restriction digestion, followed by ligation of the complementary sticky ends via DNA ligase. Each step in the creation of the construct plays a vital role, and should not go unrecognized.