Biology Case Study

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

In our experiment we are using one of three methods to inject the E.coli bacteria with

Froger, Alexandrine, and James E. Hall. "Transformation of Plasmid DNA into E. Coli Using the Heat Shock Method." Journal of Visualized Experiments ser. 2007.253 (6) Web. 22 Nov. 2016.

Bacteria are the natural world 's unsung heroes. They receive bad rep due to the disease causing individuals like tuberculosis but the vast majority of bacteria on Earth are harmless if not beneficial to both the environment and humans. Take for example E. Coli a well known bacteria that lives within our intestines. This particular bacteria makes vitamins that we need in order to stay healthy. In this experiment we analyzed the changes seen in bacteria when adding to differing DNA plasmids; pUC18 and lux. To arrive to the most accurate results possible we had to have the E. Coli bacteria made permeable to the plasmids. So it was prepared with calcium chloride. One group of bacteria was given the pUC18 plasmid, and the other group was given the lux as well as the pUC18 plasmids. We then proceeded to incubate them in different containers. Some had ampicillin where as some had none. This was done in order to observe the effect of pUC18, and lux growth patterns. A control with neither plasmid was used to keep a basis for our experiments. Bacteria that had transformed and taken in the lux plasmid without ampicilin were able to illuminate, and only bacteria that gained the pUC18 were capable of surviving with ampicillin. The results observed reveal how foreign DNA can be adapted to fit into another cell 's DNA even incorporated to be an optimal part of the cell. This process makes it a very viable manner of being able to mass replicate advantageous genes, that would

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

This is imperative in light of the fact that this is the thing that the films comprise of that makes it workable for the plasmid to enter it. Seeing how imperative a plasmid is in a life form's life is the fundamental idea of this trial do we benefit from this, as well as gives better restorative disclosures to lead a superior life. The use of vectors ensures the transportation to particular qualities that can advance improvement of anti-infection agents. Another significance of plasmids is that with further research and trials could prompt discovering the most worthwhile anti-toxins that can battle off specific sicknesses in light of the fact that they can adjust so well to situations, for example, influenza indications. Wellsprings of blunders are imperative in each trial, and in this specific one there are different. There could have been pollution from the air or water that could have gotten into the test tubes. This could have coming about because of fixing the tubes off

It was also conducted to learn about the process of moving genes from one organism to another with the help of a plasmid. The control group was the -pGlo with LB and ampicillin antibiotic, and the -pGlo with LB. The experimental group was the +pGlo with LB and ampicillin antibiotic, and the +pGlo with LB, ampicillin antibiotic, and arabinose. The dependent variable was the bacteria. It had the ability to change. The independent variable is the ampicillin antibiotic and pGlo. They stand alone and are not changed by other factors. The organism used was the bacteria GIVE TYPE OF BAC. and HOW WILL THE BACTERIA BE

The two control plates LB AMP-PGLO and LB AMP+PGLO (refer to Figure 3.0 and Figure 4.0) include a nutrient causing growth and the antibiotic, ampicillin, with or without the plasmid DNA. The purpose of the two is to provide a determination of the LB AMP ARA+PGLO and whether or not, the plasmid has an effect with the sugar. In addition, the two controls show that plasmid, pGLO, can affect the growth of bacteria with the consideration of ampicillin. As well, the two control plates prove that the results of the LB AMP ARA+PGLO and the LB-PGLO plate can be reliable knowing that they have been under the same conditions of the incubator as the control plates. Green Fluorescent Protein acts as the biological molecule for the fluorescence.

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

A 2 ml bacteria culture was picked by centrifugation at 10,000 rpm for ten minutes. The supernatant of this centrifugation was poured off and frozen until it was needed. The supernatant is resuspended by adding 250µl of Cell Resuspension Solution, to completely resuspend the pellet by pipetting. After this the tubes are only to be inverted to prevent chromosomal DNA being shared. The cells of the pellet are then lysed to release all of the intracellular contents; adding 250µl of Cell Lysis Solution does this. The pellet is incubated until the suspension is cleared. The next step could not be done until the

Plasmid was a term introduced by Lederburg in 1952 as a descriptive for genetic material that is located outside of the nucleus of a cell at points or the entirety of its replication cycle and origionates from and returns to chromosomal DNA (Lederberg, 1952). In 1968 this descriptive was adapted to being elements of the genetic material that are generally located outside of the chromosome and can carry out self-replication. recognition that it also didn’t cause cell damage which highlighted that it was different from a virus (Lederberg, 1998). The features of plasmids such as size, being extrachromosomal along with the ability of self-replication provide a basis for manipulation of genetic material alongside the ability to analyse the roles of different variations. In this report it is shown how certain techniques are carried out along with the reasoning behind the methodology to isolate and manipulate plasmids within E.coli. The initial step in the protocol was to isolate the plasmid DNA, plasmid DNA is useful when conducting experiments involving gene manipulation this is due to the determination of plasmid and chromosomal DNA. Plasmid DNA generally do not contain genes that are needed for the cells regulation (housekeeping), a genetic element that contains the genes for the cells regulation would be classified as chromosomal DNA. Due to plasmids not carrying DNA responsible for the cell to survive normal conditions they provide a gateway toward the production

Once incubation is complete the viral DNA plasmid will be extracted from the cultures using a mini-prep protocol as follows. Cells will be washed twice with PBS and lysed in lysis solution (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, and 0.2% SDS) for total genomic DNA extraction. The cell lysates will treated overnight with proteinase K (0.2 μg/ml), extracted with phenol-chloroform and precipitated with 2 volumes of 96% ethanol. DNA pellets will be dissolved in TE buffer containing 20 μg/ml RNase A and incubated for 1 hour at 37°C. The DNA will

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double