Unit 1 Plasmid Dna

Restriction enzymes are a tool that allows us to pinpoint human identity down to single differences in our DNA. Work through the following simulation so you can see these molecular scissors in action.

In this case, the DNA is the only thing left of the organism, so the process of cutting it from the source does not necessarily need to be done. In the laboratory scientists will use the process of restriction enzymes to cut the DNA into smaller parts. The cuts are made at specific nucleotide sequences. Different restriction enzymes recognize and cut different sequences of DNA. Restriction enzymes work by matching up with a space that matches the restriction enzyme. The site that it matches up with is called the recognition site. The restriction enzyme wraps around the DNA, and it causes a break in both strands of the DNA molecule. Restriction enzymes are often used in the process of DNA cloning. (Biotechnology Learning Hub,

According to Medicine.net (2012), genetic transformation is a process by which the genetic material carried by an individual cell is altered by the incorporation of foreign/exogenous DNA into its genome. Competent cells are able to accept DNA presented by experimental influence or manipulation, and the application of genetic engineering with bacteria can aid in the fight against diseases, allowing individuals to maintain their lifestyles without the threat of certain illnesses like heart disease, cancer or hereditary disorders (McPhersson 2008). Plasmid DNA are small circular double-stranded helixes, and present in the plasmid are ampr (selectable marker gene) and GFP (Green Florescent Protein), (BIO-RAD 2010).

DNA results were that of pB325 plasmid extracted from DH5α E. coli cells. Expected A260/A280 ratio was 1.8 while expected yield was ~1-3ug.

Restriction digest involves the use of restriction enzymes (also known as restriction endonucleases) to locate specific base pair sequences in DNA. These enzymes cut, or cleave, DNA only at their designated sequence, which is referred to as a recognition sequence. While there are four different types of restriction enzymes (1), the only type that was worked with in the following experiment were type II restriction enzymes (2). These enzymes have recognition sites that are mostly palindromic and usually consist of around four to eight base pairs. They also require only magnesium (Mg2+) as a cofactor to operate. Cofactors are molecules that bind to enzymes in order to activate them (3). Additionally, they cut DNA only at, or very near to, their specified restriction site, unlike other types, which cleave at various distances from their recognition site (1). The restriction enzymes that will be used in following experiment are Hind III, PVU II, and Bgl I (2). Hind III recognizes and cuts DNA at the sequence AAGCTT. It is isolated from Haemophilus influenzae (4), which is a bacteria that is the cause of several diseases, including pneumonia, and meningitis. (5) When Hind III is used to cleave DNA, the end result will have “sticky ends,” which means that there will be a few unpaired nucleotide bases on each end of

Plasmids are circular chromosomes that are found in bacteria (and other cells), can replicate independently (autonomous) and are separate from chromosomal DNA (Wikipedia, 2017). The plasmids have various conformation but the most important one is the negatively supercoiled conformation. (Wikipedia, 2017) also states that plasmids are suitable for an organism’s survival within the environment, as chromosomes generally contain all the necessary genetic information of an organism for normal conditions the plasmids on the other hand contain additional information which becomes useful in certain living

This experiment overall was very successful as using information obtained from the results of both the single and double digests, a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. For this particular experiment, a 1% agarose gel was used as this concentration ultimately results in pores that can separate the DNA by size. The process of gel electrophoresis is made possible by the electric current that is used to move the samples of DNA throughout the gel. For this particular experiment, the gel was run at a current of 100 volts. As a result of the phosphate backbone of DNA, DNA is negatively charged. Because DNA is negatively charged, it moves away from the negative electrode and moves down the gel toward the positive electrode as it is attracted that way. What allows for the resistance the fragments face when moving away from the negative electrode is the texture of the gel itself. As a result, the slower fragments of DNA have the ability to move at a faster rate than the larger fragments of DNA. Each sample loaded into the gel contains a loading-dye for two reasons. One reason is that the loading dye contains

Isolation of plasmid DNA from three cultures of E.coli using a method known as the alkaline lysis method.

RFLP or restriction fragment length polymorphism method is used in which restriction enzyme cuts the DNA at specific sites according to different restriction enzymes. Length of fragments will be different for each individual (everyone’s DNA is unique).

The motif was compared with its ancestral sequence to test, and further validate, whether it is a good binding motif. The similarities between and ancestral were compared to the ones between motif and random promoter sequences. The results showed that in most cases, a good biding motif was embedded in an ancestral repeat instead of a random promoter sequence; this confirms that the ancestral repeats were more easily able to produce a good binding

A fragment length variant that is generated through the presence or absence of a restriction enzyme recognition site

It started out as one idea, and then developed into something more valuable as many different layers began to uncover. More and more was discovered, and so it began to spread worldwide, engrossing scientists with their unique abilities. Although, it’s important to take note of the fact that a large portion of this interest began with the discovery of a plasmid to act as a vector. A vector is, simply put, a vehicle for foreign DNA. Vectors insert this genetic material into its system, and relocate elsewhere in order for the DNA to replicate and spread throughout the new location. This information proved to be crucial, for this ‘vector-like’ ability allowed for scientists to mix and match different DNA strands in order to genetically modify certain organisms. In order for scientists to accomplish this task, they must first be able to insert DNA into the plasmid vectors themselves. To do so, a restriction enzyme is used, which, depending on what restriction enzyme is selected, will cut the DNA at certain points in the base pairing sequence. This cutting process is done to both the foreign material, as well as the plasmid vector being used for transport. Once completed, both parts will have split into two “sticky ends,” which are separated pieces of the base pairings that are ready to be connected to a matching pair of DNA. It’s important scientists use the same restriction enzyme for both, in order for the same base pairings to be cut from each, leaving sticky ends that can correspond to the opposite strand of the other organisms DNA. The base pairings of each are then lined up with one another in order to create a singular strands with the separate pieces taking on the role as one singular genetic strand. This is accomplished through the help of DNA ligase, which performs as a sort of glue that bonds corresponding nitrogen bases. Once the insertion into the plasmid is completed, it is then time to

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

In Southern blotting DNA is extracted, purified, and cut into fragments with restriction enzymes. The DNA fragments are separated by size using gel electrophoresis. The DNA is transferred from the gel to a nitrocellulose filter by placing the gel on top of a sponge sitting in a tray filled with buffer. A nitrocellulose filter is laid over the gel and covered with paper towels. As the paper towels pull the buffer through the sponge, gel, and filter the DNA fragments are carried from the gel to the nitrocellulose filter where they stick tightly. The nitrocellulose filter is removed and hybridized with a radioactively labeled nucleic acid probe that tags the DNA fragments of interest. Unbound probe is washed off and the filter is exposed to X-ray film. The DNA fragments that are

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