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Dna Plasmid Transformation Lab Report

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Result DNA Plasmid Transformation GFP DNA plasmid (Vector Map) Figure 1 Plasmid map of pmaxGFP, encoding the new green fluorescent protein. MaxGFP was used as a positive control for the proper monitoring of transfection efficiency in the HeLa cell. There is comparable transfection efficiencies and mortalities in the HeLa cell that was transfected with MxGFP. HeLa cell transfected with MaxGFP usually reveals slightly higher fluorescent compared to the cells that was expressed with eGFP. This reason makes maxGFP the idea positive control protein most especially for fluorescence microscopy NFkB Luciferase reporter DNA plasmid (Vector Map) Figure 2 Plasmid map of NFkB Luciferase reporter by TNFα in HEK 293 Cells. This map works exactly as that of the GFP with slight differences. NFkB was used …show more content…

GFP DNA, bacterial transformation plate with DH5α cell. A Certain colony of bacteria grows on the surface of the bacterial plate as a result of transformation. This GFP DNA that was used in this experiment was controlled with an antibody known as kanamycin. The GFP DNA was 300 µL and its shows a rapid growth after being polymerized for 24 hours. Each of the grown bacteria includes GFP DNA in them. The plates show a clone of many bacteria that is used to perform mini …show more content…

GFP and NFkb DNA plasmid data table. The different phases that each of the DNA exhibit after their mini prep was done. The different phases that were presented in the table include DNA absorbance (A) in two wavelengths (260 and 280), their initial concentrations while it was measured in the UV spectrophotometric machine, the final concentration of each DNA, and the total concentration of each DNA that was used in the DNA plasmid transformation that was done with DH5α cell. The DNA of NFkb and GFP shows slight differences from both 260 and 280 absorbance. Also, there is about 2.000 µg/ml difference between the initial concentration of the NFkb to that of

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Results Continued: The purpose of this experiment was to show the transformation of E. Coli with pGLO. We made four different plates each with different additives to compare them to one another, and be able to track the transformation. Initially we had predicted that only one of the four plates would glow. The plate with the plasmid (+pGLO)/LB/amp/ara was the one we said would grow and glow because it contained all the necessary tools to do so.

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The plasmid and the gene are both cut using restriction enzymes. To incorporate the gene into the plasmid, both the plasmid and gen stuck together by a bacterial enzyme called ligase. The plasmid with the foreign gene can then be inserted into the bacteria One specific plasmid is pUB110 which is circular whose host is Bacillus subtilis. It has a plasmid size 2.3 kpb and copy number between 20 - 50. We can selectively grow bacteria that contain this plasmid by using selective and differential media.

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In this lab, the goal was to transform bacteria with genes that included fluorescence as well as antibiotic resistance that were taken from a jellyfish. Transformation is transferring a gene from one organism to another. Certain precautions had to be made before doing this lab since every step had to be done very quickly to prevent too much contamination. The first step in starting the transformation is to add the transformation solution into the +pGLO and -pGLO test tubes. After this is done, you put both tubes in ice and then put bacteria in both tubes.

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The hypothesis for this experiment was that transformed bacterial cells would grow on ampicillin plates and glow green when exposed to UV light. The rationale for this hypothesis was because the plasmid would code for ampicillin resistance and a green fluorescent protein, we would have the outcome explained in the hypothesis. 4. Our predictions were that for the standard protocol plates with agar and ampicillin, there would be growth and the colonies would glow under UV light. On the modified protocol on plates with agar and ampicillin where we changed the time of the heat shock, we expected less growth but the colonies would still glow.

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The reason we had the two controls were to see if we could successfully genetically transform E. Coli with the pGLO plasmid. In the beginning of the lab, we were able to predict that for the bacteria to grow, they needed the LB broth for nutrition and a gene that was resistant to ampicillin which is an antibiotic that would kill the E. Coli. In order for the bacteria to glow, they needed arabinose to activate the araC gene. After the experiment, it is safe to say that the hypothesis can be accepted. In the first dish, +pGLO Lb/amp, the bacteria was able to grow sing the pGLO plasmid was present and had the gene that was ampicillin resistant.

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Transformation in bacteria usually takes place when a bacterial cell accepts strange DNA and integrates to its own DNA. The transformation normally takes place within plasmids, which are tiny circular DNA molecules that have been separate from its own chromosome. The copies of the same plasmid range from 10 to 200 copies within a cell. These copies of plasmids may multiply when the chromosome replicate or multiply independently. One plasmid has a range of 1,000 to 200,000 base pairs.

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The morphology of the bacteria is a rod shape. Through this experiment, it can be concluded that the bacteria is a Gram negative species. The selective and differential plates were left to incubate and after viewing the results, there was no growth on either of the plates. On the MAC plate, there is a bit of discoloration of the phenol red pH indicator. Though there was a bit of discoloration, it is not a strong enough indication that the bacteria reacted with the agar because there was no growth.

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On the second day after these colonies were grown, the lab begun by labeling two test tubes -DNA and +DNA, adding the transformation solution to to each, and placing it on ice. Then the procedure called for the use of a sterile loop to pick up a colony of E coli and add it to each test tube, then after E coli was in both test tubes, the pGLO DNA was placed in ONLY the +DNA test tube. After 10 minutes on ice, and after the 4 plates are labeled each to LB -DNA, LB/amp -DNA, LB/amp +DNA, and LB/amp/ara +DNA respectively, the test tubes are heat shocked to loosen the DNA cell membrane to allow pGLO to slip in and code within the DNA. After more time in the ice bath, the tubes are taken out and set at room temperature with the LB broth placed in both test tubes. Then the dishes LB/amp +DNA and LB/amp/ara +DNA were spread with the +DNA solution, and the dishes LB -DNA, and LB/amp -DNA were spread with the -DNA solution.

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In the presence of ampicillin, E. Coli growth will not occur. Therefore, in this experiment, bacterial growth on the plate indicates the uptake of the ampicillin resistant gene into the genetic code of E. Coli. If pGLO, LB broth, ampicillin resistance, and arabinose sugar are added, then the bacteria will glow. In one of the four plates that include all of those component, fluorescence will be observed. The purpose of this experiment is to demonstrate the genetic transformation that occurs in the E. Coli bacteria.

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Transformation was successful in the plates where the bacteria consumed the pGLO plasmid. In the first plate that the bacterium was plated on it included the LB broth and of ampicillin antibiotic (amp), 2 colonies were present. The second plate of bacteria was grown with the presence of LB broth, ampicillin, arabinose sugar (ara), and 22 colonies were observed. But a green fluorescent glow of the colonies was only present in plate 2. Plates 3 and 4 were the control plates.

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In this lab we did bacterial transformation with E.coli. We performed procedures to transform bacteria into green fluorescent protein (GFP). The purpose of this lab was to find the different effects of transformation with E.coli bacteria. We spread negative and positive pGLO into four different plates to observe the different effects of each one using heat shock and incubation.

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Discussion: Since the plate represented by Figure 1 does not contain plasmid, there will not be any transformed cells. There is also no antibiotic ampicillin, therefore the cells will not die but they will grow and replicate since it is on the LB agar plate. Therefore on this plate there are only the normal E.coli cells. The LB agar plate is a favorable environment for the bacteria to grow and replicate at its optimum rate (Sezonov, 2007).

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coli strain using Ni+2Agarose Affinity chromatography column. These serial experiments were accomplished and the results were in acceptable range. The highest relative fluorescence activity was in elution fraction E3 as expected. The highest total protein amounts were found in the wash fractions as expected because non-rGFP protein was washed out and the rGFP was expected to come out in the elution fractions. The highest percentage of purity obtained was about 40% in E3 fraction, which should probably be higher with more skillful performance in the progress.

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Nucleic acid molecules are separated when placed in a chamber with an electric field that moves the negatively charged molecules through the agarose. DNA is polar since it has a highly charged phosphate backbone, making it soluble in water. Because DNA is negatively charged, it will move towards the positively charged electrode when an electric current is applied. Shorter strands move more quickly than longer strands if gel, which is why the bands are placed in order of size on the gel.

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Isolation of Bacteria For this part of the project, we were each provided with an unknown bacteria. In order to obtain pure cultures of the unknown bacteria we had to use the streak plate technique to isolate separate colonies. We used a nutrient and MacConkey agar plate to begin. We used both agars because the nutrient agar allows us to grow the largest number of different types of microbes, yet not all bacteria can grow because it is rich with beef broth and some yeast extracts.

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