Introduction to Microbial Genetics - Lab Report Example

LAB 4: INTRODUCTION TO MICROBIAL GENETICS Cell walls and membranes must be broken to release the DNA and other intracellular components trough a process called cell lysis. Therefore, lysis of the bacterial cell can also be achieved with a proper combination of enzyme called lysozyme to digest the cell wall and detergents to disrupt membranes. The ionic detergent Sodium Dodecyl Sulfate (SDS) at 80°C is used to lyse E. coli. Separation of chromosomal DNA from plasmid DNA is achieved through step 8 which involves centrifuging the tubes at 14,000 rpm for 5 minutes at 40C in a microfuge, and transferring the supernatant to fresh tubes using pipette. This process will result in separion of DNA from plasmid DNA. Protein removal is achieved through step 9, which involves addition of equal volume of phenol: chloroform to the supernatant is and mixing by vortexing, and then it is centrifuging at 14,000 rpm for 2 minutes at 40C in a microfuge to chemical denaturation. Phenol: chloroform effectively precipitates nucleic acids, whereas it is much less effective with proteins, thus allowing the removal of protein from DNA. Removal of RNA is achieved by adding solution I [50 mM glucose; 25 mM Tris HCl (pH 8.0); 10 mM EDTA (pH 8.0), 100ug/ml RNase] to the pellets as indicated in step 5. Removal of enzymes that could degrade the plasmid DNA is achieved through step 14, which involves addition of 70% ethanol at 4°C is to rinse the pellet of double-stranded DNA and centrifuging at 14, 000 rpm for 5 minutes at 4°C in a microfuge. Precipitation of plasmid DNA from solution is achieved through step 11, which involves precipitation of double-stranded DNA with 2 volumes of ethanol at room temperature and mixing by inverting 10 times. Then the mixture is allowed to stand for 2 minutes at a room temperature Purification of the plasmid DNA is achieved through step 16, which involves re-suspending the pellet in 50 μl of TE buffer by vortexing then incubating at 55°C for 5 minutes to dissolve. Conclusion The aim of the experiment was achieved in that it was possible to isolate of plasmid DNA from Escherichia coli. There were a few errors but they were minimal and negligible; such errors can be avoided by ensuring all the procedures are strictly followed and using appropriate apparatus. LAB 4B: QUANTITATION OF DNA The absorbance at 260nm to 280nm for the sample was recoded as follows: Absorbance at 260nm Absorbance at 280nm 6.653 12.702 The ratio of absorbance at 260nm to 280nm for the sample was found to be 1.91. This is a clear indication that the sample had contamination with RNA. The sample was, therefore, not pure and the RNA was the likely contaminants because contamination with RNA gives a ratio greater than 1.8. The most appropriate step that can be taken to remove these RNA contaminants is to add RNase enzyme to the sample. From the absorbance at 260nm, the concentration of DNA in the sample was found to be 635.1 mg/ml. For the Agarose Plate Technique, it was possible to get different fluorescence intensities, which allowed for determination of the approximate concentration or quantity of DNA in the sample. The approximate quantity of DNA was 2.06. The results obtained from the two methods were similar. The reason why Syber Safe added to the agarose plates used in this experiment was to make the DNA fragments visible under short wave ultra violet light. In other words, Syber Safe is used as a staining agent. Conclusion The aim of the experiment, which was Quantitation of DNA was achieved. This is depicted by the fact that the results obtained from the two methods namely ultraviolet absorption and Agarase plate were almost the same, a clear indication that the experiment was successful. LAB 5: RESTRICTION DIGESTION AND AGAROSE GEL ELECTROPHORESIS Below is the photograph of the gel containing the DNA sample Below are distances moved by the molecular markers and also by the restriction fragments of the DNA sample. molecular markers restriction fragments 36 mm 30 mm 40 mm 36 mm 45 mm 40 mm 48 mm 41 mm 53 mm 47 mm 57 mm 51 mm A graph of log molecular size of the molecular markers against distance travelled on the gel Distance (mm) Molecular size (kb) 36 10.1 40 7.1 45 5.1 48 3.1 53 2 57 1.7 From the graph above, the sizes of your restriction fragments were determined using the graph by considering the distance moved by restriction fragments checking their respective sizes. Distance moved by restriction fragments (mm) Sizes (kb) 30 9.9 36 10.1 40 7.1 41 7 47 4.8 51 0.9 How electrophoresis separates the DNA fragments and how Syber Safe enables detection of the DNA fragments. There is a specific density of gel used in the electrophoresis; the DNA is put in a well, and then a high voltage power supply is used to pull the DNA through the gel. Because spliced DNA is slightly charged, it begins to move through the gel (Hanahan, 2010). The density of the gel causes the larger pieces to go slower than the smaller pieces (Hanahan, 2010). Syber Safe is a staining agent used to make the DNA fragments visible under short wave ultra violet light. Syber Safe dyes stick to the DNA fragments making them visible under the microscope or UV light. Conclusion There exists a linear relationship between the log of mobility and the concentration of the gel over a certain range of fragment size and as a result, gel concentration must be chosen that will separate the molecules effectively in the DNA. For example, a gel of 0.8% (w/v) agarose is effective for separating DNA molecules of 0.5-10 kb in size. As a quick measure, the molecular weight of fragment of unknown size was estimated by direct comparison by eye to the position of the lambda fragments of the gel. After staining with Syber Safe, the bands are visualized by using UV illumination. Agarose gel was used to determine the purity and intactness of DNA. The less rigorous means of purifying the DNA may result in contamination of the mini-rep DNA. LAB 6: TRANSFORMATION OF Escherichia coli BY PLASMID DNA The results were recorded as follows: transformation efficiency competent no MAP competent + MAP 10-3 10-2 10-1 Undiluted Groups 2.3 * 104 + - 9 19 40 TNTC 1 7 * 103 + - 2 12 36 TNTC 2 (my group) 3.4 * 103 + - - 3 19 169 3 4.5 * 103 + - - 1 10 224 4 3 * 103 + - - 14 20 150 5 1.3 * 103 + - 1 7 65 TNTC 6 8.06 * 103 + - 2 12 116 TNTC 7 1.8 * 104 + - 1 5 92 TNTC 8 Transformation can be described as the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material, that is exogenous DNA from its environs and taken up through the cell membrane (Alberts, 2009). This may take place naturally in some bacterial species, but it can also be achieved by artificial means in some cells. Competent cells are those cells that are capable of being transformed, whether artificially or naturally (Alberts, 2009). Transformation efficiency can be described as the number of transformed cells, also known as transformants generated by 1 µg of supercoiled plasmid DNA in a transformation reaction (Alberts, 2009). It is calculated using the formular below: There are five factors affecting transformation efficiency; these include the forms of DNA, the plasmid size, growth of cells, the methods of transformation, and the genotype of cells (Alberts, 2009). Using the above formula the transformation efficiency was calculated and the answer was found to be 7 * 103. The results obtained by my group were slightly different from that of the rest of the groups. This slight difference is likely to have been caused by minor errors when caring out the experiment; such errors may include poor timing, and negligence. To detect cells that had been transformed, the mixture of treated cells was cultured on media that contain the antibiotic so that only transformed cells are able to grow and hence become visible. Cloning into the PstI site at 8.2 kb on pRY121 will lead to a recombinant plasmid that will not allow a transformed cell to be ampicillin resistant because PstI site at 8.2 kb on pRY121 specific restriction enzymes that make a transformed cell not to be resistant to ampicillin. Conclusion The experiment was successful because the aim of the experiment, which was to examine transformation of escherichia coli by plasmid DNA, was achieved. It was also possible to know various principles used in cloning processes. There were a few errors but that can be avoided by strictly following the required steps and avoiding negligence. References: Alberts, Bruce; et al. (2009). Molecular Biology of the Cell. New York: Garland Science. Becker, J.M., Caldwell, G.A. and Zachgo, E.A. (1990), Biotechnology, A Laboratory Course, Academic Press, Inc. San Diego. Hanahan, D. (2010). "Studies on transformation of Escherichia coli with plasmids". Journal of molecular biology 166 (4): 557–580. Mandel, Morton; Higa, Akiko (2008). "Calcium-dependent bacteriophage DNA infection". Journal of Molecular Biology 53 (1): 159–162. Sambrook, J., Fritsch, E.F. and Manniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbour Steffan, R.J., Goksoyr, J., Bej, A.K. and Atlas, R.M. (1988). Recovery of DNA from Soils and Sediments. Appl. Environ. Journal of Microbiology. 54 (12) 2908-2915. Read More