Replicating Genetic Cloning To Produce Red Fluorescent Protein

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

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

We were using a restrictive enzyme to cut the DNA into smaller fragments. For the restriction digest we pipet 4 micrometers of enzyme mix into the bottom of each of our colored tubes making sure to use a new tip for each sample. Next, we capped the tubes and mixed the contents by flicking the tube a little bit with our fingers. After we mixed the contents, we tapped the tube to make sure all the liquid would go to the bottom of the tube. Then, we put the colored tubes in the heating blocks and we let them incubate for 35 minutes at 37 degrees Celsius.

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

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.

For this experiment, we used two restriction enzymes called Ava II and Pvu II. Four tubes were needed for the experiment as well. Each microtubule was labeled accordingly, A, B, and C. Using Micropipettes, we added 2 microliters of pUC19 DNA into each tube. To make sure the DNA was on the bottom of the tube, we tapped each tube on the lab bench. Each tube had its own specific amount of different solutions added on, however the final volume for each tube was 30 microliters. Tube A had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Ava II enzyme. Tube B had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Pvu II enzyme. Tube C had, 23 microliters of DI Water, 3 microliters of the buffer, 1 microliter of Ava II, and 1 microliter of Pvu II. The last tube C had, 25 microliters of DI water, 3 microliter of the buffer, and no enzymes. Each solution was added according to how it is written here. After we finished adding the solutions to each tube, we tapped the tubes on the lab bench to make sure the

It’s been seen in movies like Gattaca, and now reality is catching up to fictional genetic engineering. Genetic engineering is defined as the deliberate modification of an organism’s characteristics by manipulating genetic material. Rapid progression in this field has already taken place; today, over 40% of the world’s food is genetically modified and this science has begun to allow parents to select the traits they want in their children. As of right now, genetic engineering is a fresh concept not completely accepted or understood, but by 2041, it will as common practice as smartphones are commonplace because of its contribution to the quality of life.

People always look for miracles in their life. What if there is a way to miracles? Genetic engineering can create miracles, by making impossibles to possibles. From the name genetic engineering we can define that is all about genetics. Haircolor, color skin, eye color, long or short all comes from genes that are inherited from the parents. What if we can create and customize our gene to have specific traits and actions? Genetic engineering is more beneficial than risky and has a huge effect on the today’s society because it plays a major role with treating human diseases, for pharmaceuticals and genetically modified foods for people and animals.

Genetic engineering techniques have been applied to various industries, with some success. Medicines such as insulin and human growth hormone are now produced in bacteria, experimental mice such as the oncomouse and the knockout mouse are being used for research purposes and insect resistant and/or herbicide tolerant crops have been commercialized. Plants that contain drugs and vaccines, animals with beneficial proteins in their milk and stress tolerant crops are currently being developed

In this method, cells are incubated with antibodies conjugated with fluorescent dyes such as FITC (fluoroscein isothiocyanate), which emits green wavelengths of light; PE (phycoerythrin), or Texas Red, both of which emit red wavelengths of light. Newer fluorescent dyes emit at yellow and blue wavelengths as

Then label each of them either pGLO positive and pGLO negative, depending on which one took the plasmid (4). Place both test tubes on micro-centrifuge on ice for ten minutes. Label the agar plates with three for pGLO positive and three for pGLO negative. Label the six agar plates with what they are, whether it is just LB, LB and ampicillin, or LB, ampicillin, and araC (4). There should be one for each positive and negative. After the ten minutes in the centrifuge has passed, take them out and place them in a forty two degrees Celsius heating block for fifty seconds. Once the fifty seconds have passed, transfer the tubes back to ice for two minutes. Take the micropipette and begin adding pGLO positive or pGLO negative to the correctly labeled plates. Once the pGLO is added to the agar, use the spreader to spread out the culture until it is dry on the agar plate. Make sure that before and after every use of the spreader that it is dipped in ethanol and sterilized with the Bunsen burner, to avoid contamination. Once all of the bacteria is dried onto the agar plates, use Para film to seal the agar cultures shut. Allow for it to incubate at thirty seven degrees Celsius for twenty four hours(4). After time has passed, check for growth and use an ultraviolet light to check

Genetic engineering is widely used today. Genetically altered bacteria are used to make human insulin, human growth hormone, and a vaccine for hepatitis B. Two vaccines against AIDS created with genetic engineering have begun clinical trials here in the United States ("The

Genetic transformation has the ability to alter genes, thus leading to the expression of different phenotypes. This technique can be used in a variety of sectors of biotechnology to help advance things such as agricultural production, bioremediation, and medical treatments. In this lab, we attempted to successfully complete genetic transformation on an Escherichia Coli sample, by getting the bacterium to express the green fluorescent protein, which is a protein that comes from a species of jellyfish called Aequorea victoria. The bacterial transformation method we used in this lab is made possible because bacteria take up free DNA from their environments. Bacteria also contain circular pieces of DNA called plasmids. These plasmids allow genes to be transferred more easily, and can lead to sharing of beneficial genes.

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