PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 1

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1 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 1 Practical Two: Plasmid DNA Extraction from Bacterial Cells Student s Name University Affiliate

2 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 2 Aims The purpose of the experiment was to separate plasmid from Escherichia coli by making use of the QiaPrep kit, and this process of isolation and the eventual purification of plasmid DNA from the bacterial cells depended on alkaline lysis as a principle of isolating plasmid DNA. Also, the experiment made use of a silica-based resin. After that, the DNA concentration that was present in the aqueous solution could then be acquired through a spectrophotometer; at wavelength 260nm. Principles The Plasmid DNA molecule is basically a bacterial cell containing various genes. The molecule is circular and double-stranded, and is found naturally. These genes, which are present within the plasmid molecule, have the capability of conferring the bacteria with a number of genetic benefits. This depends on genetic functionalities they possess. Native chromosomes tend to be much larger in size than the plasmids, which are in thousands of base pairs in length. Plasmids are easily distinguished from the chromosomal DNA, and they can reproduce independently, especially when they are introduced into bacterial cells. This is because the plasmids have their own replication origins. More to that, the plasmids also subsist in molecular cells as extra chromosomal genomes. Whenever a bacterium goes through the process of binary fission, then the plasmids found within the cells become replicated, hence the daughter cells tend to possess a duplicate of the plasmids. Therefore, in order to isolate the plasmid DNA from chromosomal DNA, there exist two theories. The first theory is the differentiation of the sizes of the chromosomal DNA. This is usually larger in size than the plasmid DNA. The second theory is differentiating the two DNAs in the form of renaturation and denaturation.

3 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 3 Steps and Explanations 1. The bacterial culture were centrifuged and the pellets collected: This was meant to make sure that the LB was removed, and were used in the culturing of the bacterial cells. The bacterial cells that were heavy with plasmid would deposit at the bottom. 2. The Buffer P1 was used to re-suspend the cell pellet, and the cells made sure that they did not clump together: The hypotonic buffer caused an osmotic diffusion (water molecules) from high to low concentration gradients within the bacterial cell. This in turn brought about swelling of the bacterial cells, thus making them more susceptible to cell lysis. The clumps were evaded so as to guarantee the homogenous culture was obtained. 3. The culture was incubated with the Buffer P2 (contained SDS, NaOH, RNase): the RNase was meant to degrade the RNA; the SDS dissolved the constituents of protein and also dissolved phospholipid of the cell s membrane, thus caused cell lysis where cell contents were released; finally the NaOH denatured the chromosomal DNA, the plasmid DNA and the proteins, thus stabilizing structure of the cell membrane. 4. Buffer N3 was added, for a rapid neutralization of the lysate: The salt potassium acetate precipitated SDS alongside the chromosomal DNA, cellular debris, and denatured proteins. The Plasmid DNA renatured and stayed in the mixture. This is because the plasmid is small in size and covalently closed. The co-precipitation of insoluble compounds enable the isolation of the wall-bound chromosomal DNA from the plasmid DNA. 5. The precipitated debris was removed by centrifuging them, and loading the clear lysate: The negatively charged plasmid DNA attached the surface of resin, which is positively charged. Other substances were in the flow via fraction. The Wash buffers were then used to entirely get rid of all the residual contaminants.

4 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 4 6. The Buffer EB was added to try and elute the plasmid DNA: Here, the buffer disturbed the interaction(s) between the plasmid DNA and the resin, and this became ready for use for further experiments. Results Wavelength Absorbance Value 260 nm nm For the calculation of the extracted DNA the formula below was used: 900μl of water was diluted with 100μl of DNA, as the dilution factor being; = ( )/100 = 10 Concentration of the extracted DNA = by 50 by 10 = 189μg per ml Actual DNA yield = 189μg/ml by 0.1ml = 189μg Purity of the DNA = A260/A280 = 0.378/0.198 = Discussions Proteins and organic compounds are commonly known are common contaminants of the nucleic acids. Five nucleotides constitute the RNA and DNA, and they portray numerous

5 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 5 ratios of A260/A280. It is possible to evaluate the purity of the nucleic acids, by calculating the absorption ratio at the wavelengths 260nm and 280nm. From a hypothetical perspective, a sample of a pure DNA is supposed to have a ratio of A260/A280 that s roughly 1.8 to 2.0. On the other hand, a pure RNA sample is supposed to be about 2.0. Therefore, lower ratios meant that the result indicated a protein contamination. RNA always indicates higher readings than the DNA because of its higher Uracil content, which is more than that of Thymine in DNA. Because of the interactions of UV light, ring systems and the pyrimidiness, the nucleic acids are capable of absorbing light at a stronger wavelength of 260nm. Proteins such as tyrosine, phenylalanine and tryptophan have the tendency of absorbing light at wavelength of 280nm. This means that light absorptions of wavelengths 260 and 280 are normally employed so as to measure DNA contamination. From the results carried out in the experimental phase, it was indicated that the DNA sample was not as pure as expected, with a ratio of 1.909, which was within the theoretical value that is between 1.8 and 2.0. Because the change of the ph was rapid and fast, it meant that the time for pairing of the DNA strands was insufficient, thus the strands couldn t pair correctly. Therefore, the bases paired with bases on different strands, or the bases paired with other complimentary strands. This caused the DNA strands to entrap with each other, hence leading to the straps being precipitated out. The DNA substances that were present at the time, within the whitened pellet, at the end of the tubing, knowing that was following the Load N3 was added. Both DNA and the RNA were negatively charged because of the phosphate backbone within the nucleotides, which were also negatively charged. Hence, the RNA and the DNA had to bind in order to be positively charged with the resin and get it elated when the buffer was applied.

6 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 6 Since both the RNA and the DNA absorb light at wavelengths 260nm, it means that this allows for quantification of the nucleic acid concentration. However, there is a downside to this kind of quantification. Contaminants like the RNA, proteins and genomic DNA are capable of exhibiting some sort of observance at wavelengths 260nm. In case the experimental procedures were not carried out in the most appropriate and effective manner, the presence of the culture would have led to 260nm or higher. For example, the absorption of phenol at 280nm would lead to an upset of the ratio value. This is because phenol has an absorbance that is strong at a wavelength 270nm, and it possesses a ratio of 2.0. With the contaminants in place, the concentration of DNA in the sample meant that it might be an overestimate, thus a downstream process may be affected. To be able to improve on this experimental part, you should subject the DNA test at different values from the wavelengths. The absorbance values connected with 230nm might imply that the DNA was contaminated with Chaotropic salts and/or normal compounds. For case, the Guanidium salts tend to help the DNA regarding binding with silica-based articles, which absorb light at 230nm. Should this value boosts, then it means that the attentiveness of salt covered is lesser when compared with expected. With this ratio known, the volume of salt within the sample is usually determined. In theory speaking, the ratio of absorbance of the pure nucleic acid that has been measured at wavelengths 260 and 230 tends to be between 2.0 as well as 2.2. This type of range of values is somewhat greater than the ratio regarding absorbance measured in wavelengths 260 as well as 280. The presence of the contaminants would lower the ratio value to lower than While doing so, the absorbance sizes at a wavelength of around 320nm indicate your existence of turbidity within the sample, thus may pose a chance of a likely toxic from the

7 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 7 sample. This means the in relation to measures of absorbance regarding sanitized DNA biological samples from 230 to be able to 320, this gives results that will be more reliable. A better alternative method that is going to measure concentration of DNA without worrying about contamination is perhaps utilizing the Agarose Gel Electrophoresis. This method uses a dye often known as Ethidium bromide, which often quantifies the DNA. In essence, the concentration of your DNA can be calculated by researching its band high intensity to molecular weight markers for your already known DNA concentrations. Certain contaminants just like the genomic DNA as well as RNA can as well be noticed through the Agarose gel approach. That is, for your genomic DNA, it will be noticed at the top of the gel as it slowly migrates, because high molecular bonds. On the other hand, the RNA will be at the bottom in the form of your smear, due the low molecular bonds. From the results, the actual DNA yield was estimated to be 20.2µg, which was a closer in value to the theoretical value of 20µg. Nevertheless, the A260/280 ratio was found to be 2.26, which proved that the DNA was not as pure as expected. In other words, the ratio proved that the 20.2µg contained some contaminants. Therefore, to improve on the yield s purity, it is advisable to make sure that all the cells are lysed. This is to ensure that the contents within the plasmid DNA are all released; to the maximum. At the same time, the volume of Buffer N3 is also important that it is increased so as to guarantee that every DNA is eluted out of the resin column. During the step of purification, it is also imperative that the tip of the pipette does not touch the pellet when transferring the supernatant into the QIAprep the supernatant contains plasmid DNA. You have to do this since the insoluble pellet may hamper using the plasmid with regards to binding the DNA to the silica based articles. This reduces yields on the plasmid DNA that is being eluted since many of the plasmid may not have the capability to bind to the resin s surface, thus would seem within the flow-through fraction.

8 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 8 Further discussions (Cuvettes techniques) A lab tissue was used in cleaning the cuvette before putting it in the spectrophotometer: This was to make sure that the cuvette s surface was clear of fingerprints and any other unwanted material. Hence, it contributed to a higher accuracy and a better reliability of the measured samples. It was made sure the clear side with the Cuvette faced the correct side of the spectrophotometer: This is for the reason that absorbance is commonly measured by the volume of light that experiences the sample. However, inadequacies within the cuvette may have caused some inconsistencies in terms of readings, especially when the cuvette may not have been positioned in the correct manner. That is, it may not have been positioned in the same manner for every measurement being tallied. During measurements under UV light, the Quartz cuvettes were more preferable than the plastic cuvettes. The reason for this was because plastic cuvettes tend to absorb UV light very strongly, which in turn tend to interfere with the overall results. Unlike the plastic cuvettes, the Quartz cuvettes demonstrated an absorbance for the Ultraviolet.

9 PLASMID DNA EXTRACTION FROM BACTERIAL CELLS 9 References Dale, J., & Schantz, M. (2012). From genes to genomes concepts and applications of DNA technology (3rd ed.). Oxford: Wiley-Blackwell. Extraction from Bacterial Cells. (n.d.). Grange, J., & Fox, K. (2009). Genetic Manipulation Techniques and Applications. Chichester: John Wiley & Sons. Plasmid DNA Extraction from Bacterial Cells. (n.d.). Retrieved from Silhavy, T., & Berman, M. (1984). Experiments with gene fusions. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.

Practical Of Genetics

Practical Of Genetics To learn how to extract plasmid DNA from E.coli and to observe the analysis of plasmid DNA by gel electrophoresis. Over the past decades it became evident that virtually in all bacterial