DNA Extraction from Microbial Cells
DNA of an organism is very vital in determining the genetic information of an organism. In order to carry out study of DNA, it must be extracted from the original cells of an organism. This paper outlines the use of spin column method in extracting DNA from E.coli. The experiment was conducted in the lab using the provided reagents and equipment.
Aim of the experiment
The main aim of this experiment was to use spin column technique to extract genomic DNA from E.coli.
Genes play a very vital role in determining the makeup of organism. They are also essential in determining the ability of an organism to survive in the environment as well as its function. DNA (Deoxyribonucleic acid) is known to contain the macromolecular components of cells that has genetic information of the organism. It is the content of DNA that assist in classification of organisms as well as determining the capability of the organisms. In order to know more about an organism, DNA is extracted and then analyzed (Lutz, Sweedler and Wevers, 2013, pg.76).
There are several techniques that can be used in extracting DNA. Generally DNA extraction involves the process of biomass concentration, cell lysis, debris removal and finally nucleic acid precipitation. It is also important to know the quantity and quality of DNA present in the sample that need to be used for other analysis such as DNA sequencing, cloning as well as PCR.
One of the DNA extraction method is spin column technique. This technique to be able to produce more DNA that is more pure without the use of organic solvents. The first step of this method is lysis which involves cell wall breakdown to expose the cell content such as nucleic acid, proteins, salts and lysate. The breakdown of bacterial cell wall is achieved by use of lysozyme enzyme. Letting the proteins free is ensured by use of Proteinase K enzyme. After lysis, lysate is altered by changing its chemical condition to allow the DNA precipitation from the entire solution (Zhou and Ling, 2011, pg.34). DNA get bonded in the filter column as lysate passes out. DNA is then harvested by washing it using buffer. Elution of DNA involves changing chemical condition of the DNA by using a solution to make it a solution. The figure 1 below gives the pictorial view on how the process takes place.
Apparatus and reagents
In order to conduct the experiment, the following apparatus and reagents were provided; Micro centrifuge, vortex, collection tube, column, microfuge tube, Tris-EDTA (TE) buffer, BTL buffer, proteinase K, BDL buffer, HBC buffer, wash buffer, elution buffer, scissors and paper towel.
All the sample tubes provided were first dried up at the start of the experiment using paper towel. Cell pellet were put in a microfuge tube. 100 μL of TE buffer was added to the microfuge tube containing cell pellets. The tubes were then vortex to re-suspend the cells. 10 μL of Lysozyme was added to the cell and later flicked gently in order to mix them. For 10 minutes, the tube was incubated at 370C. 100 μL of BTL buffer and 20μL of proteinase K was added to the cells and incubated for 20 minutes at 550C. During incubation, at 5, 10 and 15 minutes, the tubes were removed and flicked to mix and returned to the water bath. 220 μL of BDL buffer was added to the sample. The sample was then incubated for 10 minutes at temperature of 650C. 220 μL of ethanol was added to the sample. The sample was then pipetted up and down in order to thoroughly mix it. A column was then inserted into 2ml collection tube and sample was transferred into the column. At 10,000g the sample was centrifuged for one minute. The flow was then discarded and column returned back to collection tube. 500 μL HBC buffer was added to the column and centrifuged for one minute at 10,000g. The flow was then discarded and column returned back to collection tube. Lid was removed from the tube and microfuge labeled by use of scissors. After placing column into the tube, 30 μL of elution buffer was added. The tube was then incubated at room temperature in a period of 5 minutes. Using a balanced centrifuge, the tube was spin for one minute. The column was discarded. The sample was then transferred to a clean microfuge and lid closed. The sample was labeled as DNA with date and name of the student noted. The sample was then stored in a freezer for one week.
Principle and Theory
Spin column provides a simple and efficient way of extracting DNA up to 200ug. The lysis process is carried out producing lysates which are then cleared (Butler, 2005, pg.234). The spin column selectively absorbs plasmid DNA. The impurities such as salt, and nucleotides are washed away.
Results and discussion
From the experiment the DNA sample was extracted successfully. The extracted DNA was kept safely in a period of one week waiting for the concentration estimation. The sample of DNA that was extracted was used in Agarose Gel electrophoresis.
Protein is a very important is operation of the cells making the organism. In order to understand the function of the cell and its content there is great need to analyze protein. There are several methods that are used in estimating the concentration of protein as well as its size. This paper discusses the process of estimating the protein concentration by use of both Warburg-Christian method and the BCA assay method. Warburg-Christian method was used to calculate the absorbance value of the four samples associated with standard curve and measuring absorbance value. By use of BCA method, the respective concentration of four samples protein were put on test for further analysis. The results obtained from the two methods are further compared and discussed to come up with a more superior method.
The main of the experiment was to use to Warburg-Christian method and the BCA assay method in detecting proteins and estimate proteins concentration.
Cell is a basic unit of life. It is noted that huge part of cell is formed by proteins. Proteins play important roles in the cell. Some of the functions include transportation function, catalysis function as well as roles in structure. In order to study and understand cells with its functions, proteins should be studied in details. Study of proteins requires it extraction from the cells. Purification of proteins requires one to know the amount of proteins present in sample. In order to estimate the concentration of proteins in a sample an assay is used (Uversky and Dunker, 2012, pg.28). Absorbance of a solution when UV spectrophotometry is used plays a greater role in determining protein concentration. The two commonly used method in protein concentration estimation are Warburg-Christian method and (Bicinchoninic acid) BCA assay. It is observed that Warburg-Christian method is dependent on absorbance of UV light at 280 nm that is done by aromatic amino acid side chains. BCA assay method on the other hand involves the combination reduction of Cu2+ to Cu1+. This is done by alkaline medium that has sensitivity to the detection of cuprous cation by the bicinchoninic acid (BCA) (Kamp, Calvete and Choli-Papadopoulou, 2004, pg.321). Bioengineering provides the required tools necessary for exploiting protein analysis.
Dilution was done to prepare three samples of standard proteins having range of concentrations. Standard dilution of an aliquot was pipetted in the cuvette. By use of the spectrometer AS260nm and A280nm for the sample in the cuvette were read against the buffer blank. The result was used to plot a graph in order to determine the relationship between concentration of the standard protein and absorbance measurement. The unknown concentration of the proteins in the samples was estimated.
Bicinchoninic acid (BCA) method
Dilution was done to prepare three samples of standard proteins having range of concentrations. 300µ1 of reagent B was added to the 15ml of reagent A in order to prepare protein assay. The tube was inverted to ensure mixing up of the content. Each sample was prepared in a group of three. 25μL of assay was added to each of the protein samples and the container labelled as 7ml bijou container. For each of the container having samples, 500µl of the BCA protein assay was added (reagent A+B). The content was incubated for 20 minutes at 370C. After the incubation period the color change was noted and optical absorbance of both samples and standard sample read at 562nm. The graph was then plotted to draw the relationship between the absorbance of the standard protein and the one measured (A562nm). The concentration of each protein was then estimated.
Principle and Theory
Warburg-Christian method basically rely on the relative absorbance of both proteins and nucleic acids that takes place at 280mn and 260nm. It is noted that tyrosine and tryptophan that are found in proteins get absorbed at 280nm. This method is best suited for semi quantitative analysis of protein samples due to that fact that the amount of tyrosine and tryptophan vary from protein to protein. Bicinchoninic acid (BCA) method has high sensitivity to the extent that as low as 1 μg, protein can be detected. The reduction reaction takes place due to the presence of nitrogen in peptide bond in proteins and the reaction is temperature and alkaline condition dependent (Walker, 2009, pg.92). The following formula is used in calculating protein concentration when Warburg-Christian method is executed.
Results and discussion
Data for the experiment were recorded as follows.
|Protein standard volume ml
|Water volume ml
In order to assess the unknown proteins in UV light at 260nm and 280nm, the results were tabulated as follows.
Using BCA method, the results were tabulated as follows.
|Sample volume μl
|BCA volume μl
At A562nm, the results were as follows
Looking at the graph the two data that were obtained from the experiment showed some linearity although the graph was not straight.
The ratio of the two methods is not 1:1. This is because it can be seen that 0.3 from the BCA corresponds to 2 in Christian Warburg method.
Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260
Other methods like nuclear magnetic resonance can be used in assessing comparability.
Source of error can be use of contaminated apparatus leading to inaccurate results. Human error during measurement can also lead to inaccurate results.
Concentration (mg/ml) = (1.55 x A280) – (0.76 x A260)
From the two results it is observed that BCA assay is more accurate that Christian Warburg. This is because its results is closer to the expected value.
Other method that can be used is Bradford assay due to the fact that the reagents used are not affected by presence of the reducing agents.
Several methods can be used in carrying out protein extraction and quality assessment. However method of extraction plays an integral role in determining accuracy in concentration as well as the amount of protein that is isolated. From the results obtained from the two methods BCA assay proves to be a better method. However, there is need to employ better technique in protein isolation as well as concentration estimation.
Agarose Gel Electrophoresis
The main objective of the experiment was to use the Agarose gel electrophoresis in assessing the quality of DNA that was extracted in the previous lab practical and estimate the quantity of DNA.
In order to understand genes and their relationship, it is essential know how DNA quality assessment is carried out. One of the important technique that is used in DNA concentration analysis is Agarose gel electrophoresis. It is based on the effect of electric field on the molecules in a solution due to that fact the molecules has charge on them. The separation and speed of movement of molecules depend on charge and size. It involves the application of the electric field to solution that contains proteins or DNA. Application of electric field to a solution results in movement of the molecule or DNA in a speed and direction that is dependent on the size and charge of the molecules (Kurien and Scofield, 2012, pg.321).
During the DNA quality assessment, agarose gel acts as the matrix. In the process of using agarose gel, samples of DNA are placed near the cathode before application of electric field. When electric field is injected, molecules moves to the anode side.
The technique of agarose gel electrophoresis is very much important when there is need of quantifying small amount of DNA. This technique is superior to other techniques in that it allows the visualization of DNA and other nucleic acid is possible. This technique involves the use of sample from unknown DNA and that of known DNA (standard). The two samples are run in the agarose gel and then stained through the use of ethidium bromide (Kurien and Scofield, 2012, pg.61). The UV rays helps in viewing the stained bands. After running the samples, the intensity of the unknown DNA bands are compared to the one of the standard DNA determine the concentration of unknown DNA. In order to obtain accurate results, keenness and precision are priorities that must be considered. Figure 1 below gives the pictorial view of the experiment.
Apparatus and reagents
Agarose gel, test tubes, centrifuge tubes, measuring cylinder, DNA sample and timer
Using the extracted DNA and water, three solutions were prepared by diluting extracted DNA in the centrifuge tubes. Caution was taken to ensure that the resulting solution was 10L. After dilution, 10L of undiluted and diluted DNA samples were loaded on the agarose gel. 5L of 1kb standard DNA was also loaded in the agarose gel. Agarose gel was put on and set at 120volts. The gel was then run for 40 minutes to allow movement of the DNA molecules to the positive terminal. Upon completion of the running process, the dye was allowed to move three quarter way along the gel. Thereafter it was taken to UV light allow image taking. The quantity of DNA was then estimated.
Principle and theory
Electrophoresis is a method that is used in separation molecules that have net charge. The molecules move in a differential speed when electric field is applied. From the Principe of magnetism, it is observed that like charges repel while unlike charges attract. DNA molecules has negative charge therefore move to the anode when exposed to electrophoresis. The speed of movement depend on the charge, molecular size and molecular weight of the DNA molecules (Kurien and Scofield, 2012, pg.65). For this reason several bands of DNA molecules are formed after electrophoresis process is completed. It is noted that molecules with small size and less weight will move faster as compared to molecules of large size due to reduced friction during motion. Agarose gel used is obtained from seaweed and is suitable for DNA separation as it allows the visualization of DNA fragments.
Results and discussion
In order to calculate the concentration of the unknown DNA sample, the concentration of the standards DNA was used.
Other methods that can be used in estimation of DNA concentration are absorbance methods and Fluorescence Methods.
DNA sample from gel electrophoresis method can be kept for further use in downstream process. It allows easy visualization of the DNA sample. Agarose proves to be one of the best method used in DNA separation due to its ability to handle low percentage of gel in separation of large DNA fragments. It was observed that the higher the concentration of the gel the smaller the pore size.
Lutz, N., Sweedler, J. and Wevers, R. (2013). Methodologies for metabolomics. New York, NY: Cambridge University Press.
Zhou, C. and Ling, X. (2011). DNA binding and DNA extraction. Hauppauge, N.Y.: Nova Science.
Butler, J. (2005). Forensic DNA typing. Amsterdam: Elsevier Academic Press.
Uversky, V. and Dunker, A. (2012). Intrinsically disordered protein analysis. New York: Springer.
Kamp, R., Calvete, J. and Choli-Papadopoulou, T. (2004). Methods in proteome and protein analysis. New York: Springer-Verlag.
Walker, J. (2009). Basic protein and peptide protocols. Totowa, N.J.: Humana Press.
Kurien, B. and Scofield, R. (2012). Protein electrophoresis. New York: Humana Press.