DNA isolation is a very important technique which forms the basis of many types of techniques such as diagnosis of many genetic diseases as well as genetic fingerprinting DNA. Regardless of the quantity and purity required, the type of DNA is what makes the difference between different DNA isolation methods. There were three different cultures of E. coli whose aim was to analyze the DNA of E. coli. Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get the original essay Several techniques have been used to manipulate and isolate DNA from E. coli. coli. We begin by isolating the plasmid DNA from the 3 cultures by alkaline lysis. Alkaline lysis is an extraction method used to isolate plasmid DNA from bacteria. Then the DNA isolated by the restriction enzymes is used for digestion. Restriction enzymes are capable of cleaving DNA and transforming it into fragments, within the molecule, at sites called restriction sites. Bacterial transformation was also carried out. Finally, analysis of transformed E. coli is performed using agarose gel electrophoresis. Practice 1 Three cultures of E. coli A, B and C were provided and were grown overnight with shaking at 37°C. The bacterial pellet is dissolved in 100 ml of solution 1. Solution 1 contains 50Mm of glucose, the purpose of glucose is to ensure an osmotic balance between the cell and the solution and this prevents the cells from bursting at this stage. Solution 1 also contains 25 m m Tris (pH 8.0), used to stabilize the pH of the solution. 10Mm EDTA is also a chemical necessary to allow enzymes to degrade DNA. The main purpose of EDTA is to bind to magnesium and calcium, which prevents DNA degradation. EDTA is also capable of stabilizing the phosphate backbone of DNA as well as the cell wall. The following solution II is added, solution II is composed of 0.2 M NaOH and 1% (w/v) SDS. This strongly alkaline solution is capable of disrupting cell membranes and allows plasmid and chromosomal DNA to come into contact with and denature. The contents of the cells came into contact with the extracellular chemicals, allowing the EDTA to chelate with the metal ions present in the cells. SDS precipitates with proteins in the cellular contents and forms an insoluble complex. As a result, precipitates were observed in the solution. Chromosomal DNA and plasmid DNA were denatured by the high pH of the solution. The process is known as denaturation because it then adds solution 3 which is 3M potassium acetate pH 4.8. Potassium acetate is able to decrease the alkalinity of the solution, which allows it to renature plasmid DNA but does not renature chromosomal DNA. Double-stranded DNA can renature double-stranded DNA because the hydrogen bonds between single-stranded DNA are restored. Through hydrophobic relationships, a white precipitate is formed by the SDS, denatured cellular proteins, and single-stranded genomic DNA stuck together while the double-stranded plasmid dissolves in the solution. At this point, most of the cellular debris is separated from the plasmid DNA but the solution contains the debris, salts, Rnase and EDTA, so the solution must be cleaned and the plasmid DNA concentrated. 70% ethanol is then added and it is able to change the structure of the DNA as it aggregates and precipitates from solution. Through centrifugation, the precipitated DNA can be separated.Practice 2From E. coli isolated, we then begin to degrade the DNA using restriction nucleases. THERestriction enzymes cut DNA molecules in specific areas to cut them into smaller fragments. Different types of DNA sequences are cut and recognized by different restriction enzymes. These restriction enzymes also need a suitable buffer that includes magnesium as a cofactor. A certain concentration and a Tris to buffer the ph. For different types of enzymes, there are different optimal salt concentrations. Samples B and C are isolated with 10 units of enzyme. There are 2 tubes called BR tube and CR tube. The BR tube contains B DNA, 10 x EcoR1 buffer, EcoR1 enzyme and water, the CR tube contains the same but instead of B DNA it contains C DNA. There is a specific order in which these are added. First, water is added, then it's the buffer, the DNA and finally it's the enzyme. The reason for this order is that a suitable environment must be created before the enzyme is added. Eco R1 is essentially a restriction enzyme isolated from E. coli, which at particular locations cuts the DNA double helix at a specific restriction site. EcoR1 is able to cut the backbone of both strands, resulting in two sticky ends at the DNA cut site. There is a specific sequence that EcoR1 can recognize. This sequence is GAATTC and the enzyme cuts between the G and the A on the strand which is complementary. To start, the solution is added in an orderly manner with water, buffer and enzyme. Water is used to dilute the buffer because the manufacturer concentrates the buffer. The EcoR1 buffer is there because it is the optimal buffer used for enzyme performance. When conditions are finally suitable for the enzyme, it is added. This is when it opens or fragments the plasmid DNA. Once both tubes were finished, they were incubated. Plasmid transformation. Now a technique known as bacterial transformation is used and two tubes B and B are diluted in Tris buffer to pH 7.6, which is 40 times the final volume of the mixture, as they have been diluted they are labeled diluted B and diluted BR. The goal of bacterial transformation is to introduce DNA into bacterial cells. There are many techniques used to achieve this, but the reliable technique is the thermal shock technique. Once the DNA is absorbed, it must either attach to the host genome or replicate autonomously. Circular forms of DNA are the only ones that can replicate; linear forms that use restriction enzymes will not be able to transform. The circular shape when introduced into E. coli will be able to replicate. The heat shock technique uses calcium chloride which creates a calcium rich environment, between the plasmid DNA and the bacterial cell membrane, the calcium rich environment cancels the electrostatic repulsion between them. In the bacteria, pores are created following a sudden increase in temperature, which allows the entry of plasmid DNA into the bacterial cell. When the cell takes up the DNA, it settles down to create a stable transformant. In practice, the unknown strain of E. coli cells was added with calcium chloride and pre-cooled in ice. The procedure is repeated twice and kept on ice. At the same time, tube 1 which does not contain plasmid DNA is prepared, tube 2 containing diluted B plasmid is prepared, and tube 3 containing diluted BR plasmid is prepared. Pre-cold competent cells were added to tubes 1, 2, and 3 and mixed gently. Since the cells are fragile, it is important to avoid using the vortex. After 15 minutes, the tubes were shocked with hot water at 42°C. At this point, the cell membrane thinsand the plasmid DNA can enter the cell body. After 2 minutes, the tubes were placed on ice for 5 minutes to allow the cell membrane to recover. L of broth is added to each tube and bathed in water at 37°C for at least 20 minutes. After that, cells from each tube were transferred to LB amp plate and incubated overnight. Practical 3 agarose gel electrophoresis is a commonly used method to analyze the size, purity, quantity and sequence of DNA molecules and plasmid DNA molecules. Agarose is a polysaccharide, it is one of the components of agar and is extracted from red algae. It is also made up of anhydrous galactose units. There are many reasons why agarose gel is beneficial for gel electrophoresis. Between the non-covalent bonds of the polysaccharide unit are formed by the agarose gel. A sol state forms as noncovalent bonds to maintain the structure of the agarose gel so that it undergoes a phase transition at high temperatures. When the running buffer and agarose powder are mixed, it creates the gel with subsequent arrangement of the ground state at a higher temperature and also organized cooling. The agarose gel is prepared by pouring the melted agarose into it first. Wells are formed in the DNA sample for the DNA to be loaded by combs, they are then left to harden for approximately 20 minutes. Once the gel is set, the TBE buffer is used to carry a current and deliver ions, it is also able to maintain the Ph. As we know, DNA is negatively charged, so when the electric field is applied during the electrophoresis period, there will be movement. of the DNA towards the anode which is the positive pole. The sample loading wells are oriented toward the negative pole which is the cathode, so when the gel is placed in the electrophoresis tank it is oriented. A loading buffer solution is used to treat the plasmid sample before it is loaded onto the gel. The density of the sample is increased because the loading buffer contains glycerol. DNA is able to move toward the positive electrode because the larger fragments are slowed down compared to the smaller fragments, which is why they do not travel far. A band also forms when all the fragments gather at one point and move at more or less the same speed. So now when all the fragments have traveled and separated the fragments of different sizes, there is a dye known as SYBER-SAFE and this is used to visualize the DNA. When the dye is exposed to UV radiation, an orange-colored fluorescent light appears. Finally, trans UV lighting photographs the gel that contains the colored DNA molecules. A loading buffer completes the circuit and balances the pH in the gel. Results The transformed E. coli from tubes 1, 2 and 3 were cultured in the agar plate. Tube contained 0 colonies, tube 2 contained 300 colonies, and tube 3 contained 5 colonies. Tube 1 contained no colonies as it only contained buffer. Tube 2 contained 300 colonies because it contained circular plasmid DNA. Tube 3 only contained 5 colonies because it only contained linear DNA, so the only way it could have contained colonies was possibly through contamination or mutation. From the results, tube C shows that it did not travel a long distance because the molecules were large or had a low molecular charge and therefore could not pass through the gel network. Incomplete precipitation of chromosomal DNA could be a possible error. We know that tube A does not contain a plasmid.