Restriction Digest Lab Report

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

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).

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

The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well

The goal of the restriction digests is to be able to cut the plasmids at specific sites. This step

Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.

· Describe how a piece of foreign DNA can become a part of a plasmid that ends up altering the final phenotype of cells

Gas Production in Relation to Combustion Reactions Introduction Chew, swallow, digest, and pass: these may not seem like anything other than simple functions, but they are very important when it comes to the process of digestion, as well as, the environment. This is because all of these actions relate to the subject of gas production. Reactions in which gasses are formed take place in many aspects of the natural world. Whether it is within the atmosphere or within the body.

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

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.

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

Recombinant bacteria is a bacterium cell that has had its genome changed, by the alteration, or edition of a gene. Recombinant DNA is DNA that has been artificially prepared by cutting DNA into smaller fragments and joining fragments together, that would not normally be found together naturally- even fragments taken from different organisms.( Principles of Gene Manipulation sixth edition, S.B. Primrose, R.M. Twyman and R.W. Old ) DNA can be "cut" using the enzyme Restriction endonuclease, which recognises a specific sequence of base pairs- called recognition sequences (Genetics Fourth Edition- Hugh Fletcher, Ivor Hickey). These enzymes are secreted naturally by bacteria as a defence mechanism. The enzyme cuts between the bases within this sequence in a "staggered pattern", leaving what are known as "sticky ends", which are off branches of single-stranded DNA (http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html). A vector, such as a plasmid in the case of a bacterium, is then cut with the same restriction enzyme, DNA polymerase is then used to add nucleotide bases by complementary base pairing to the sticky ends from two different cut strands. DNA ligase can then join these two strands into a single strand of rDNA (http://www.ncbi.nlm.nih.gov/books/NBK21881/). This vector, usually a plasmid can then be taken up by bacteria cells (transformation) becoming part of their genome. After it is taken up by the host cell, the plasmid is able to replicate, which

General safety instructions followed during lab experiments comprising wearing lab coats, safety glasses, and disposable gloves. Also, no open toe shoes allowed in the lab. Generally, pipetting skills are important through all experiment especially for Bradford assays because the absorbance reading very sensitive to bubbles and so the concentration of the samples. Whereas in SDS-PAGE experiment, preparation and fast demonstration is very important to avoid solidifying the gel before transfer it to the cassette. In mammalian gene expression experiment, sterile condition is highly important to evade mammalian cell infection. As previously mentioned mammalian cell grow slowly and yield low protein, hence infection will result in reduced protein expression.