Using Information Obtained From The Single And Double Digests

10. Which had the greatest average 1- OD (amount of bile acid adsorbed to meal particles), the standard meal with bile acid or the

Let’s follow the path of a delicious ham and cheese sandwich with lettuce and pickles as it is eaten and digested! Start at the beginning and discuss the anatomical parts as well as the biochemical roles that contribute to this sandwich being turned into chemical energy. Be sure to include mechanical and chemical mechanisms, along with how they are metabolized in the body!

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

DNA. The isolated plasmids are ran on 40 ml 1% agarose gel at 110 volts with 1.2 μl 6X track

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.

When choosing the restriction enzymes to use, many factors are considered such as methylation of certain genes, which can influence the results of the experiment. Methylation will protect the gene from restriction enzymes, so in this experiment restriction enzymes that are not methylation sensitive are used (3). The wavelength of the UV Radiation can also have an impact on the rate of mutagenesis (4), which will be discussed more in the results. Restriction enzymes in this experiment such as Bam will cleave every six base pairs at a specific location on the target site (5). Bacteria such as E. Coli can naturally make restriction enzymes such as Eco, which is used naturally by the host cell to cut viral DNA (typically from phages) and make it non-functional in order to protect the integrity of its genome (6). In this discovery-based experiment, we will observe the changes in banding patterns of E. Coli DNA with multiple restriction endonucleases after UV induced mutation, and use these results against the control to determine if there are any changes in gene sequence by the number of base pairs in the observed DNA

Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA

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

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

1. What is peristalsis? 2. What are the peristalsis and what are they used for? 3. What are the three salivary glands and where are they located? 4.

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

The uncut plasmid does not have a linear shape instead it adapt different conformation including supercoiled and open nicked DNA (also known as open circle or relaxed) conformation. Contrastingly, the linear conformation that appears once the DNA cut using restriction enzymes. Different DNA conformations reveal different velocity in agarose gel. The tightly coiled the DNA the faster it will travel in the agarose gel. Simply, the supercoiled DNA conformation will travel easily through agarose mesh where the relaxed DNA conformation will travel slowly through the agarose mesh due to the physical hindered. The lowest speed correlate with the linear DNA conformation being the most conformation that hold back by the agarose mesh. The uncut plasmid presented in agarose gel (see figure 2) was not treated with any restriction enzymes (in lane 1 please refer to table2) and shows two

Recombinant plasmid pAB2 was isolated from a culture of E. coli, this contained an insert of baculovirus. The restriction enzyme called restriction endonuclease was used to cut the circular plasmid DNA, allowing us to identify which fragment has successfully been cloned from the insert of baculovirus. The agarose gel electrophoresis was performed using Gelled in order to utilize the DNA in gel, this binds to the DNA and fluoresces under UV light.

Each one recognizes a unique sequence of 4-8 nucleotides in a DNA molecule and are cut at specific locations within the site. The cutting is done by the use of restriction endonucleases, also called enzymes. Hundreds of restriction enzymes have been identified. “A bacterium carrying such a plasmid is able to live and multiply in the presence of an antibiotic, while cells lacking the plasmid are unable to divide. Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities.” (A Kuspa, WF Loomis) In the following experiment a recombinant plasmid was created. A restriction enzyme was chosen based on the presence or absence of a gene (LacZ). The LacZ gene codes for an enzyme called beta-galactosidase.

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