Competent E. Coli Lab

In this case, our plasmid DNA is isolated from a liquid culture of the E.coli that was transformed. This is done by reacting the liquid culture with five buffers. The buffers are P1, P2, N3, PE, and an elution buffer. P1 is used to re-suspend the pellet and degrade RNA, P2 is used to lyse cell membrane, PE is used to wash the sample, the elution buffer is used to release DNA from the spin column, and N3 is used to precipitate proteins and genomic DNA. The main components of P1 are Tris, EDTA, and RNase. The main components of P2 are NaOH and SDS. The main component of PE is ethanol and the main component of N3 is acetic acid. The main component elution buffer is water. The possible contaminations of mini-prep are proteins and salts (Garey et al.,

As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew

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

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

Both tubes were incubated on ice for 15 minutes. The cells were removed from the ice and immediately “heat shocked” by putting the tubes in a hot water bath of 42 degrees Celsius for 90 seconds. After 90 seconds, the tubes were put back on ice for another minute. 250 microliters of Luria Broth (LB) was added to both tubes by using a transfer pipette. The luria broth acts as a source of nutrients for the E.coli, enabling the E.coli to grow. The solutions were mixed by tapping the tubes with a finger. The cells were incubated at 37 degrees Celsius for 5

E.coli was the chosen bacteria for this type of experiment. It is a gram negative bacterium that will grow rapidly given ‘any culture medium with the necessary energy source, nutrients, pH, and temperature’. Therefore, MacConkey Agar being the medium for its growth will enable us to achieve the experiment

In the following experiment, Escherichia coli were transformed using six separate agar plates. Transformation occurs when plasmid DNA is uptake into the bacterial cell. The two ways transformation is facilitated is by placing them in calcium chloride (CaCl2) and heat shock. These two methods allowed the bacterial cells to become competent or more open to the uptake of plasmid DNA. The purpose of this experiment was to transform bacterial cells with plasmid containing ampicillin resistance and lux genes. It was hypothesized that the lux genes would have created a light-emitting reaction on the bacteria while the ampicillin genes should have made the bacteria resistant to ampicillin attacks and be able to grow normally. The experiment results

There are five stages to this experiment: pre-incubation, incubation, heat shock, recovery and growth, and selection. First, two micro test tubes were obtained and labeled “+” and “-”. These labels were representative of transformed (+) and untransformed (-) Escherichia coli (E. coli). 250 microliters of transformation solution (calcium chloride) was added to the test tubes and placed on ice. During the pre-incubation period a single colony was placed into the test tube labeled “+” and was completely dispersed in the solution. This was repeated for the tube labeled “-“ as well. Then the solution was returned to the ice. In the incubation period, the pGLO DNA solution was added to the tube labeled “+” but was not added to the tube labeled “-“. The tube was then returned to the ice and incubated for 10 minutes. While this was undergoing the process, four agar plated were labeled according to what they had been treated with. The plates were treated accordingly: plate 1 contained lysogenic broth (LB)/ ampicillin (amp)/ plasmids with a green fluorescent protein (pGLO); plate 2 contained LB/amp/arabinose sugar with pGLO; plate 3 contained LB/amp without pGLO; and plate 4 contained LB without pGLO. The next step was heat chock. Both test tubes were placed in a water bath that was set at 42°C for 50 seconds. After 50 seconds the test tubes were rapidly transferred back to the ice were they

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

Bacterial transformation is one of the main ways scientists are genetically engineering cells. The reason why bacteria is getting used for these experiments is because bacteria’s genes are very versatile. For the purpose of this experiment we used the bacteria Escherichia Coli, the reason for this is that this specific bacterium is resistant to antibiotics. Plasmids were used in this experiment for the purpose of identifying what type of growth will occur also because plasmids are needed in order for transformation to be possible. Plasmid is not the only thing needed, also needed are vectors and a host. The bacteria used, E-Coli, served as the host in this experiment and the plasmids served as the vectors. Vectors are used to deliver foreign

The purpose of this experiment was to study the transfer of genetic information on plasmid F’lac by using Escherichia coli. Plasmid transfer was measured by using two different methods. The first one was by using selection and contraselection with three antibiotics: streptomycin(which was replaced by naladixic acid for the second part of the experiment),ampicillin and kanamycin and the second one by using a colour indicator ( X-gal). As significant results, the percentage of transfer for F’lac was higher than the percentage for transposition. Also, the experiment demonstrated that E.coli can quickly acquire resistance to several different antibiotics through the transfer of the F’lac plasmid. It was concluded that significant

In this experiment students attempt to transform E. coli with a plasmid. The plasmid that will be used to transform the bacteria in this experiment is pGal and occurs naturally in E. coli. Transformed bacteria cells will grow in the presence of the antibiotic while bacteria cells that did not take up the plasmid, will not grow. Students will use mathematical calculations to determine the efficiency of the transformation process. The transformed E. coli with the

Plasmids are small DNA atoms that are found in the nucleus region of a bacterium. They provide an extra assistance to a bacterium that allows them to survive and reproduce in certain environments, such as making the bacteria resistant to specific antibiotics. In the experiment being conducted, plasmids pUC18 and lux will be tested to determine the different types of growth with Escherichia coli present. In order to do this, CaCl2 was transferred into a tube with E.coli and incubated for 10 minutes. Different measurements of micro liters of various substances are added to the test tubes and later incubated for 15 minutes. A separate test tube is collected, labeled and added its corresponding micro liters of the competent cells. Once this is

A plasmid vector must have (1) an origin of replication to allow it to be replicated in E. coli independently of the chromosome, and (2) a selectable maker , usually antibiotic resistance. The selectable maker allows the presence of the plasmid to be easily identified through selection, cells that contain the maker will live when plated on antibiotic –containing growth media, and cells that lack the plasmid will not live (they are killed by the antibiotic). A fragment of DNA is inserted by the techniques described in figure 16.2 in a region of the plasmid called the multiple cloning site (MCS).

After being transformed into bacterial system and grow overnight, the mutated plasmid needs to be isolate from the system which is based on the different denatured stage of bacterial genome and mutated plasmid DNA (3). With this step, the first centrifugation allows the separation of the growth media and bacterial cells; the added lysis solution is able to denature the bacterial genome and mutated plasmid DNA; the neutralization solution is