Ligation is the process of joining two DNA fragments or other molecules by a phosphate ester linkage with the action of enzyme. Ligation of DNA is process that involve the DNA of interest is inserted into the plasmid. This 3’-hydroxyl of DNA terminus are joined together with 5’-phosphoryl of another by phosphodiester bond. The ligation of DNA fragments usually performed by using T4 DNA ligase. All the reaction components such as ligation buffer, DNA insert, pGEM®-T, and T4 DNA ligase is mixed by gentle pipetting. The 2X rapid ligation buffer is used in this experiment to saving the ligation time. This ligation buffer contains ATP. ATP is used to catalyze the formation of phosphodiester bond and the reaction of restriction enzyme buffer. The recommended time and temperature of incubation when using 2X rapid ligation buffer are one hour at room temperature (24°C) and overnight at 4°C. In this overnight incubation at 4°C is applied to achieve maximum number of recombinants.…
Calcium Carbonate is found naturally in food products. It is needed for everyday, common body activities. Calcium Carbonate is used to prevent Calcium deficiencies. Some common names for Calcium Carbonate in the medical industry are Tums, Alka-Mints, and Maalox. Calcium Carbonate has other uses besides medical purposes, such as building materials and construction, and paper, plastics, paints, and coatings. You can also find it in chalk, limestone, and marble. Calcium Carbonate is able to be extracted from marble in a pure form. It is also able to be prepared when Carbon Dioxide is put through Calcium…
7) In gene cloning, the bacterial cells take up the recombinant plasmid DNA through a process called transformation. Bacterial cells can be transformed using electric pulsation or heat. The short electric pulse or a brief rise in temperature causes openings in the plasma membrane. The bacterial cells make copies of the recombinant plasmid DNA during cell…
When a bacterium integrates a piece of DNA into its genome, bacterial transformation has occurred. In this experiment bacterial transformation will be done using calcium chloride/heat shock. This is done by incorporating the plasmids into chemically competent cells that were made permeable by the calcium chloride solution and heat shock. In 1928, Frederick Griffith, a physician from London, was he first person to experiment with bacterial transformation. He permanently transformed a safe, nonpathogenic bacterial strain of pneumococcus into a deadly pathogenic strain. [1]…
.2400 grams of the unknown compound. This is done in duplicate and purple-tinted precipitates are placed in Gooch crucibles. The precipitates are suction dried using ethyl alcohol then acetone to…
This experiment is a continuation of the lab’s efforts to purify L-LDH. Previously our enzyme was purified through anion-exchange chromatography on Q-Sepharose, but to get the enzyme out of the stationary phase NaCl was added to salt it out. However salting out the enzyme prevents using chromatography because of NaCl’s high ionic strength. Dialysis was used to remove the salt from the enzyme sample, when put into a membrane bag which uses principles of equilibrium to remove the salt via nanometer sized holes in the membrane bag which allow for water and small molecule transport. However, dialysis must be done twice, as not all the molecules of salt will leave the dialysis bag because dialysis uses Le Chatelier’s principle equilibrating the bag. The top of the bag has an air pocket introduced to allow for floatation which prevents the stir bar from damaging its contents.…
In order to find the formula of the copper chloride hydrate, we had to separate the compound to find the mass of water and copper. To begin this process, we evaporated the water and created an anhydrous compound, meaning we were left with only CuxCly. By calculating the weight of both the anhydrous and the hydrated compounds, we could conclude that the difference in the weights was the weight of the H2O. From this we were able to calculate the percent composition of CuxCly and H2O (see Calculations: Part I: Dehydration of Hydrated Copper Chloride), which showed how much of the compound was copper chloride by mass. Since there are only two stable forms of copper chloride, we concluded that possible formulas would be either CuCl or CuCl2. Intending…
Use calcium hypochlorite if you need to add calcium into the pool water. It needs to be pre-dissolved before it is used to shock the pool.…
In the experiment, we tested a sodium chloride solution. Along with the tested solution, control groups (water and sodium phosphate) were used to be help understand whether or not NaCl was a buffer. Water was the negative control group and sodium phosphate was the positive control group. If NaCl was a buffer than the pH would be stabled as the sodium phosphate buffer. If NaCl was not a buffer than the pH would fluctuate like the negative control, water. During the first trial and prior to the drops of 0.5 M of HCl acid, the pH of sodium chloride was 7.50. After the addition of 5 drops of 0.5 M of HCl, the pH decreased by 4.83 and ended at 2.67 on the pH scale. When comparing the results of the sodium chloride to the control groups, the total pH change of sodium phosphate was only…
The following experiment method is based on the procedure given through the Biology Department at UWM (Wimpee, 2006). This experiments started with two tubes of 100 uL E. coli cells, labeled one and two. Tube one just contained normal E. coli cells. Tube two was the tube with the plasmid added to it. The first step in this experiment was to add plasmid DNA, the “mini chromosomes” of the bacteria, to the E. coli cells in order to change the genetic makeup of them. I then added 10 uL of the plasmid to tube two. The next step was to chill both the tubes E.…
PLASMID DNA ISOLATION, RESTRICTION ENZUME DISGESTION AND AGAROSE GEL ELECTRIPHORESIS Abstract: The gel is covered with an ion- containing buffer, such as (TAE), that controls the pH of the system and conducts electricity. overall DNA concentration was lower than expected. Using agarose gel electrophoresis is to separate and visualize the DNA fragment, which is produced by restriction enzymes . Introduction:…
This lab was performed to see the Transformation abilities of, Escherichia coli into ampicillin resistant bacteria. For this lab, a total of six agar plates were used for growing the bacteria. Transformation occurred when plasmid DNA goes into the bacterial cell via a vector. The two ways change is encouraged is by setting them in calcium chloride (CaCl2) and applying the process of heat shock. These two strategies permitted the bacterial cells to end up capable or more open to the uptake of plasmid DNA. The reason for this analysis was to change bacterial cells with the plasmid containing ampicillin resistance and lux qualities. It was theorized that the…
Purpose: The Purpose of this lab is to utilize, demonstrate and understand the various techniques and procedures used to gravimetric labs. For this particular lab we will utilize our scientific knowledge of related to gravimetric procedures to find the chloride content in an unknown soluble salt.…
2) The same amount of DCPIP was added in each test-tube (1 cm3) using the same 5cm3 syringe…
The main application of plasmids is as cloning vectors in gene cloning. In gene cloning, a fragment of DNA, containing the gene to be cloned is inserted into a circular molecule called the “vector” to produce recombinant DNA molecule. Plasmids are one of the most commonly used “vectors” for this purpose. They transport the gene into a host cell, such as a bacterium, which is said to be transformed with the recombinant molecule. Here, these plasmid vectors multiply, producing numerous identical copies of itself and the gene that it carries. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division; they replicate independent of the chromosomal DNA. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell. Thus after a number of successive cell divisions, various identical host cells are produced, thus, the gene carried by the plasmid is “cloned” (Brown, 2006; Lodish et al., 2000). Furthermore, the transformed bacterial host cells may have the ability to express the gene, thus producing proteins encoded by the gene included in the recombinant plasmid. This application can be used for producing proteins in large quantities which can be purified further, as is done for the production of recombinant insulin. Also, plasmids could be used in gene therapy, for delivery of therapeutic gene of interest into human host cells, without causing cell injury, oncogenic mutations and an immune response (Daugherty, 2007; Lipps, 2008).…