Introduction:
In this lab experimentation, the group conducted Deoxyribonucleic acid isolation and restriction analysis on a plasmid from Escherichia coli cells. Plasmids are small circular DNA that are in the bacterium cells. Escherichia coli is a gram- negative bacterium that is known for variable reaction to antibiotics, and can be genetically manipulated. The gram- negative bacterium, Escherichia coli can be genetically manipulated by extracting a certain plasmid that allows it to resist ampicillin. If a bacterium is carrying a plasmid that encode for antibiotic resistance, it will grow and become reproductive in the environment of that antibiotics. The plasmid denoted as ‘pAMP’ contains the gene that encodes for ampicillin resistance. …show more content…
Almost half of the solutions had to be on ice to slow down their reaction. The materials that the group had were pAMP/MM294 overnight culture; Tris/EDTA (TE); Ethanol- EtOH 95 percent, and Isopropanol – IsoP. These are the most important components of the DNA isolation process. This experiment starts by extracting E. coli/ pAMP and transferring 1 ml of the resuspend culture into three 1.5 ml microtubes by using the P1000 pipette. While finishing that up, each group used the centrifuge and spun the samples for 1 minute. The centrifuge is a machine that rotates at a rapid speed that can separate fluids of different densities. Through this process, a pellet appeared at the bottom of each tube. Also with the pellet, there was a supernatant, and that was disposed of in a waste container afterwards. To get the pellet, the group had to invert the tubes slowly and gently and remove rest of the fluid inside each tube. After having the pellet isolated, 100 µl of Mini prep 1 that was on ice into each tube. Contents of Miniprep solution are 50 mM of glucose, 25 mM Tris-Cl of pH 8.0, and 10 mM EDTA of pH 8.0. With these contents being added into each tube, the glucose helps to maintain an osmotic pressure, and the Tris will buffer the cell of pH 8.0. The role of the EDTA in this solution is to bind to the cations in the lipid bilayer, which weakens the cell’s envelope. At this rate, the EDTA limits the DNA from degradation. After adding this solutions, the group resuspend each tube by vortexing each tube. Vortexer is a machine that mixes small samples of liquids. Adjusting the pipette to 200 µl, we add the second Miniprep solution or MP 2. This solution contains 0.2 M NaOH, and 1 percent of SDS. This solution lyses the cells. SDS is a detergent that dissolves the lipids that are contained in the cell membrane and the cellular proteins. While NaOH will denature both the chromosomal and plasmid DNA into
In this lab experimentation, the group conducted Deoxyribonucleic acid isolation and restriction analysis on a plasmid from Escherichia coli cells. Plasmids are small circular DNA that are in the bacterium cells. Escherichia coli is a gram- negative bacterium that is known for variable reaction to antibiotics, and can be genetically manipulated. The gram- negative bacterium, Escherichia coli can be genetically manipulated by extracting a certain plasmid that allows it to resist ampicillin. If a bacterium is carrying a plasmid that encode for antibiotic resistance, it will grow and become reproductive in the environment of that antibiotics. The plasmid denoted as ‘pAMP’ contains the gene that encodes for ampicillin resistance. …show more content…
Almost half of the solutions had to be on ice to slow down their reaction. The materials that the group had were pAMP/MM294 overnight culture; Tris/EDTA (TE); Ethanol- EtOH 95 percent, and Isopropanol – IsoP. These are the most important components of the DNA isolation process. This experiment starts by extracting E. coli/ pAMP and transferring 1 ml of the resuspend culture into three 1.5 ml microtubes by using the P1000 pipette. While finishing that up, each group used the centrifuge and spun the samples for 1 minute. The centrifuge is a machine that rotates at a rapid speed that can separate fluids of different densities. Through this process, a pellet appeared at the bottom of each tube. Also with the pellet, there was a supernatant, and that was disposed of in a waste container afterwards. To get the pellet, the group had to invert the tubes slowly and gently and remove rest of the fluid inside each tube. After having the pellet isolated, 100 µl of Mini prep 1 that was on ice into each tube. Contents of Miniprep solution are 50 mM of glucose, 25 mM Tris-Cl of pH 8.0, and 10 mM EDTA of pH 8.0. With these contents being added into each tube, the glucose helps to maintain an osmotic pressure, and the Tris will buffer the cell of pH 8.0. The role of the EDTA in this solution is to bind to the cations in the lipid bilayer, which weakens the cell’s envelope. At this rate, the EDTA limits the DNA from degradation. After adding this solutions, the group resuspend each tube by vortexing each tube. Vortexer is a machine that mixes small samples of liquids. Adjusting the pipette to 200 µl, we add the second Miniprep solution or MP 2. This solution contains 0.2 M NaOH, and 1 percent of SDS. This solution lyses the cells. SDS is a detergent that dissolves the lipids that are contained in the cell membrane and the cellular proteins. While NaOH will denature both the chromosomal and plasmid DNA into