DNA isolation is a very important technique that forms the basis for many types of techniques such as the diagnosis of many genetic diseases and DNA fingerprinting. How much quantity and purity is required, the type of DNA is what makes the difference between different DNA isolation methods. There were three different E. coli cultures, the goal was to analyze the E. DNA. coli. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay Numerous techniques have been used to manipulate and isolate Escherichia coli DNA. We begin by isolating plasmid DNA from the 3 cultures by alkaline lysis. Alkaline lysis is an extraction method used to isolate plasmid DNA from bacteria. Subsequently the DNA that has been isolated by restriction enzymes is used for digestion. Restriction enzymes are able to break down DNA and transform it into fragments and this happens within the molecule at sites called restriction sites. Bacterial transformation was also carried out. Finally, the analysis of E. is carried out. coli that is transformed and this is achieved using agarose gel electrophoresis. Practice 1 Three cultures of E. coli A, B and C were provided and grown overnight with shaking at 37°C. The bacterial pellet is dissolved in 100 ml of solution 1. Solution 1 contains 50 Mm glucose. Glucose aims to provide an osmotic balance between the cell and the solution and this prevents the cells from bursting at this stage. Solution 1 also contains 25 mm Tris (pH 8.0), used to stabilize the pH in the solution. EDTA 10Mm is also a necessary chemical to enable enzymes to degrade DNA. The main purpose of EDTA is to bind to magnesium and calcium and this prevents DNA degradation. EDTA is also capable of stabilizing the phosphate DNA backbone and cell wall. Solution II is then added, solution II is 0.2 M NaOH and 1% (w/v) SDS. This strong alkaline solution is capable of disturbing cell membranes and allows it to come into contact with and denature plasmid and chromosomal DNA. The contents of the cells came into contact with extracellular chemicals which allowed the EDTA to chelate with the metal ions in the cells. SDS precipitate with proteins in cellular contents and form an insoluble complex. As a result, precipitates were observed in the solution. The chromosomal DNA and plasmid DNA were denatured by the high pH of the solution. The process is known as denaturation because solution 3 is added next, which is 3M potassium acetate pH 4.8. Potassium acetate is able to decrease the alkalinity of the solution therefore it is able to renature plasmid DNA but does not renature chromosomal DNA. The ssDNA can renature the dsDNA because the hydrogen bonds between the single-stranded DNA are re-established. Through hydrophobic relationships, a white precipitate is formed consisting of SDS, denatured cellular proteins, and single-stranded genomic DNA sticking together as the double-stranded plasmid dissolves in the solution. At this point most of the cellular debris is separated from the plasmid DNA but debris, salts, RNase and EDTA are present in the solution, so the solution must be cleaned and the plasmid DNA concentrated. Subsequently, 70% ethanol is added which is capable of modifying the structure of the DNA as they aggregate and precipitate from solution. Using centrifugation, the precipitated DNA can be separated.Practice 2From E. coli isolate we then begin to degrade the DNA using restriction nucleases. The enzymes ofrestriction cuts 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 require 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 DNA B, 10 x EcoR1 buffer, EcoR1 enzyme and water, the CR tube contains the same but instead of DNA B it contains DNA C. There is a specific order in which these are added. First the water is added, then you get the buffer, the DNA and finally the enzyme. The reason for this order is because a suitable environment must be created before adding the enzyme. Eco R1 is basically a restriction enzyme isolated from E. coli, which cuts the DNA double helix at particular places at a specific restriction site. EcoR1 is able to make cuts in the backbone of both strands and this allows for 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 A on the strand which is complementary. To start the solution is neatly added with water, buffer and the enzyme. Water is used to dilute the buffer because the manufacturer concentrates the buffer. EcoR1 buffer is present as 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 completed, both tubes were incubated. Plasmid Transformation Now a technique known as bacterial transformation is used and two tubes B and B are diluted in a Tris buffer at pH 7.6 and this makes up 40 times the final volume of the mixture, as they have been diluted they are labeled as diluted B and BR diluted. The purpose 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 absorbed, the DNA must join the host's genome or replicate on its own. Circular forms of DNA are the only DNA that will be able to replicate, the linear form that uses restriction enzymes will not be able to transform. The circular shape once 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. Pores are created in bacteria as there has been a sudden increase in temperature so this allows the plasmid DNA to enter the bacterial cell. When the cell absorbs DNA, it settles down to create a constant transformant. In practice, calcium chloride was added to the unknown strain of E.coli cells and pre-cooled on ice. The procedure is repeated twice and kept on ice. At the same time, tube 1 containing no plasmid DNA is prepared, tube 2 containing the diluted B plasmid is prepared, and tube 3 containing the diluted BR plasmid is prepared. Pre-chilled competent cells were added to tubes 1 2 and 3 and mixed gently. Because cells are fragile, it is important to avoid the use of vortexing. After 15 minutes the tubes were shocked with hot water at 42°C. At this stage the cell membrane becomes thinner and plasmid DNA can enter the cell body. After 2 minutes the tubes wereplaced on ice for 5 minutes to allow recovery of the cell membrane. L of broth is added to each test tube and soaked in water at 37°C for at least 20 minutes. The cells in each tube were then transferred to the LB amplicon plate and incubated overnight. Practical agarose gel electrophoresis 3 is a commonly used method for analyzing 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 also consists of anhydrous galactose units. There are many reasons why agarose gel is useful for gel electrophoresis. Non-covalent bonds are formed between the polysaccharide units via the agarose gel. A sol state is formed by noncovalent bonds maintaining the structure of the agarose gel so that it undergoes a phase transition at high temperature. When the running buffer and agarose powder are mixed, the gel is created with subsequent arrangement of the sol state at higher temperature and also organized cooling. To start an Agarose gel is prepared by pouring melted agarose into the first. Wells are formed in the DNA sample for the DNA to load onto the combs, then they are left to sit for about 20 minutes. Once the gel has been solidified, the TBE buffer is used to carry current and supply ions and is also able to maintain the Ph. As we know, DNA is negatively charged, so when the electric field is applied for the period of 'electrophoresis there will be movement of the DNA towards the anode which is the positive pole. The sample loading wells face 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. Sample density increases because the loading buffer contains glycerol. The DNA is able to travel to the positive electrode because the larger fragments are slowed down compared to the smaller ones, which is why they don't travel far. A band also forms as all the fragments gather in one place and travel at more or less the same speed. So now, when all the fragments have traveled and separated the different sized fragments, there is a dye known as SYBER-SAFE and this is used to visualize the DNA. When the dye is hit by UV radiation, an orange fluorescent light is produced. Finally, UV transillumination photographs the gel containing the colored DNA molecules. A loading buffer completes the circuit and balances the pH in the gel. Results Transformed E. coli from tubes 1, 2, and 3 were grown in the agar plate. Tube contained 0 colonies, tube 2 contained 300 colonies, and tube 3 contained 5 colonies. Tube 1 contained 0 colonies as it only contained swab. Tube 2 contained 300 colonies as it contained circular plasmid DNA. Tube 3 only contained 5 colonies as it only contained linear DNA, so the only way it could have contained colonies could have been through contamination or mutation. From the results, tube C shows that it did not travel far as the molecules were large or had a low molecular charge, so they were unable to pass through the gel network. Incomplete precipitation of chromosomal DNA could be a possible error. It is known that tube A contains no plasmid but tubes B and C contain plasmid. This is because the results show that there are 2 DNA bands on both tubes B and C but there are no DNA bands in tube A. The plasmid DNA that.