Annotated Bibliography On Plasmid Purification

The instructor split the class into two separate groups one with the plasmid lux and the other with the plasmid pUC18. Group two was assigned to test the lux plasmid. The, Eppendorf, tubes were labeled “C” for the control plasmid DNA and “lux” for the plasmid lux DNA. The two tubes were then placed into the ice bath. Using a sterile micropipette 5 uL of the lux plasmid was added to the tubes labeled “lux” or 5 uL of the control plasmid was added to the tubes labeled “C” for the control plasmid DNA. Eppendorf tubes had 70 uL of the competent cells added to them with a different transfer pipet. All the tubes were then stored in the ice bath for about fifteen minutes. Another test tube was labeled “NP”, which stands for “No Plasmid”, and 35 uL of competent cells was added to each of the test tubes labeled “NP” during the fifteen minutes. Once the fifteen minutes are up, all three tubes were placed into a preheated water bath at 37 °C for about five minutes. To both the lux

Bacterial plasmids differ from bacterial chromosomes by not being essential to bacterial growth and metabolism. They are extracellular chromosomes that contain DNA. Bacterial chromosomes are essential for bacterial survival.

After that, a new sterile loop was used to immerse the pGLO plasmid DNA stock tube into the +pGLO tube but no into the – pGLO tube. Both tubes were well closed and put back on the ice for 10 minutes. While both tubes were sitting on ice, the four plates were labeled. LB/amp plate was labeled +pGLO, LB/amp/ara plates were labeled +pGLO, LB/amp plate was labeled – pGLO, and LB plate was labeled – pGLO. After that, the tubes were transferred into a water bath set at 42° C for exactly 50 seconds, then placed rapidly back on the ice for another two

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.

A single colony of bacteria was then added to each tube using a sterile loop. Using a new sterile loop, a small amount of pGLO plasmid DNA was obtained and mixed into only one of the centrifuge tubes. Each tube was then placed into an ice bath. After ten minutes, both tubes were placed into a 42°C water bath for precisely 50 seconds and then immediately placed back into the ice. After another two minutes on ice, the tubes were removed and placed on a rack.

Fill the rest of the column with NaCl, and do so by pouring the solution along the interior side of the column. This allows the gel to become moist.

Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own

In experiment of Bacterial plasmid DNA extraction, solution 1 which consists of ice-cold resuspension buffer helped corrupting the cell membrane. Because cell membrane has not been broken down yet the collected blend is clear. However, later on inclusion of Solution 2 which is lysis buffer the cell membrane was broken. The solution 3 that is a neutralization buffer was added and it provided the contaminated genomic DNA to precipitate. Using centrifugation method provided the plasmid DNA to isolate from cell debris and genomic DNA. Protein contamination was tried to prevent using ethanol.

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

Plasmid DNA is an autonomously replicating circular DNA molecule that is only about 1,000-20,000 base pairs in size and is separate from bacterial chromosomes. Plasmids are a significant part of molecular biology because they are small enough to be cloned or modified, they endure self-replication, they are stable over long periods of time and they have a diverse range of gene expression. Having these characteristics allows plasmids to introduce genes into bacteria. The difference between genomic and plasmid DNA is that genomic DNA is DNA that is part of an organisms chromosomes, while plasmid DNA is a small loop which is separate from the organisms chromosomes.

Plasmids were extracted using the three-step alkaline lysis procedure as outlined by Kado and Liu, 1999. The extracted plasmids were then digested using 1 U of each XhoI and XbaI, respectively according to manufacturer’s (New England Biolabs) recommendation and ran on a 1% Tris Acetate EDTA (TAE) agarose gel. 1 μl of BioLine Hyperladder I was used as the DNA marker.

Plasmids have many desirable characteristic which includes being easy to work with, self- replicating, stable and functional in many different species. Plasmids are circular DNA molecules which range in the size between 1-400 kb. Plasmids are easy to work with because of their size. They are easy to modify with the cloning technology available today and because they are so small they have small DNA sequences. Plasmids are the basis of recombinant DNA technology. They can produce enzymes that can degrade antibiotics. Inserting new DNA is also easy due to the plasmids ability to be cut open easily without falling apart and they can potentially make endless copies. Smaller plasmids tend to have higher copy number and can have tens or even hundreds of copies per chromosomes. Plasmids characteristics make them very promising for the development of

An alkaline lysis procedure was performed and followed by binding to a silica gel filter. The lysis released plasmid DNA from the cells and the purification on the binding membrane separate the plasmid DNA from bacterial cell suspensions. There is a possible error in this section that can lead to contamination of plasmid DNA with chromosomal DNA. Vigorous stirring can force chromosomal DNA to be in the supernatant along with plasmid DNA. Therefore, mixing during the lysis procedure should be carried out by only gentle inversion. For the second part of this section, the DNA concentration was determined using a nanodrop spectrophotometer. The purity, contamination, and absorbance were also found along with the DNA concentration. All

Plasmids are small circles of DNA, which carry additional genes and are used to exchange DNA between bacterial cells. DNA is always circular, and not associated with any proteins to form chromatin, it carries the genes for the proteins the cell needs. Prokaryotic cells do not have membrane bound organelles, like

Plasmids are small, circular, DNA molecules within a cell. They are not connected to the chromosomal DNA of the cell, which means that they are essentially free-floating in the cytoplasm. They contain only a few genes and their own ‘origin of replication’-a stretch of DNA that ensures that the plasmid is replicated by its host. A plasmid is an interesting part of the cell as it only contains DNA that codes for a specific function that is not necessary for the day-to-day survival of a cell, but only during certain particularly stressful times, but during the remaining times they still manage to not be discarded by their host cell.