The isolation of Plasmid DNA using the alkaline lysis method
Mini-prep method
Mbali Sibuyi 4121058 Biotechnology 222
Practical coordinator: Sindisiwe Nene Demonstrator: Jaime
Due: 17/03/2023
Abstract
Circular non-chromosomal DNA plasmids, which are most frequently seen in prokaryotes, include genes that increase the likelihood that organisms will survive. The genes contained in plasmids are helpful in many different ways and occasionally they contain an important gene that must be accessed for cloning or gene therapy. Without harming or shearing it, the
plasmid would need to be separated from the rest of the cell.One way to achieve this is through alkaline lysis, which entails the lysis and denaturation of the cell and chromosomal DNA. Three solutions are used in alkaline lysis: resuspension, lysis, and neutralizing solution. The lysis solution, which contains NaOH and SDS and is responsible for the
bacterial lysis as well as the denaturation of chromosomal DNA, is given to the bacteria after it has been treated with the resuspension solution to maintain the osmolarity of the cell.
Finally, the bacteria is given the neutralizing solution, which precipitates the genomic DNA along with cellular proteins and SDS. A clear solution with a few contaminants was in the end tube.
Introduction
Plasmid is a small, circular, extrachromosomal DNA molecule that is capable of self- replicating and it is found in prokaryotes and some eukaryotes (Andreou,2013). Plasmids are small in size because they only carry genes that enhance an organism's survival, such as antibiotic resistance. Plasmids aid in conjugation, the metabolism of nutrients, and the transmission of information from one organism to another in prokaryotes, and they are frequently used as cloning vectors, used to prepare recombinant DNA, and gene therapy (Bashkirova ,2005). In order to obtain and use plasmid, it must be isolated from the rest of the cell, and alkaline lysis is a technique that can be used to achieve this. Alkaline lysis involves lysing the bacterial cells, denaturing the DNA and proteins, neutralizing the solution, and extracting the plasmid DNA (Guruatma 2010). Alkaline lysis is used to isolate and extract plasmid DNA from bacterial cells during this miniprep procedure. Using the alkaline lysis method, the aim of the experiment is to isolate and extract plasmid DNA.
Materials and Methods
The experiment began with the collection of bacterial cells via centrifugation in a microfuge for 60 seconds. The supernatant liquid was decanted from the eppis tubes, which were then placed in a rack for five to ten seconds. 100 microliters of suspension solution were added to each of the two tubes, and they were resuspended by shaking until the bacterial pellet
dissociated. Each tube received 200 microliters of lysis solution, which was mixed by inverting the tubes. After two minutes, the tubes were allowed to stand for another 60
seconds. A white precipitate can be seen after adding 150 microliters of neutralizing solution to the tubes and inverting them several times. The tubes were centrifuged at full speed for five minutes. New sterile tubes were introduced and treated with 250 mL of isopropanol. The centrifuged tubes were removed and the supernatant decanted with a 1mL pipette. The liquid was transferred to the new tubes, where it vortexed for five to ten seconds before being centrifuged for 30 seconds at high speed, yielding a white pellet. After decanting the supernatant, the pellets were washed with 750 microliters of 70% ethanol. The tubes were vortexed briefly before being centrifuged for 60 seconds. The ethanol was decanted, and the tubes were centrifuged for 30 seconds before being allowed to airdry for 5 minutes. After dispensing 50 microliters of TE, the tubes were resuspended and kept at 4 degrees Celsius.
Results
After centrifuging the bacterial cells, a brown pellet was observed, which contained the bacterial cells. After adding 150 microliters of the neutralizing solution, a white precipitate was observed, which was the chromosomal DNA; however, the plasmid DNA remained suspended in isopropanol when the liquid was transferred to the new tubes. The plasmid DNA solution can be seen as a clear substance with a few impurities at the end of the experiment.
Discussion
The plasmid is seen as a clear solution at the end of the procedure andit dcontains a few impurities. The addition of the resuspension solution to the bacteria cells, which is composed of glucose, Tris, and EDTA, maintains cell integrity. EDTA chelates divalent cations in the solution, protecting the plasmid from DNases and assisting in cell wall destabilization whilst
the glucose keeps osmotic pressure constant, preventing cells from bursting (Guruatma 2010).
The lysis solution consists of sodium hydroxide and sodium dodecyl sulfate, The double- stranded DNA in the cell and your plasmid is converted to single-stranded DNA by the action of NaOH, which also aids in the breakdown of the cell wall, this is referred to as denaturation (Bashkirova 2005). The neutralizing solution caused a portion of the solution to precipitate as a white substance, which is made up of denatured proteins, genomic DNA, and SDS. The rest of the solution contains the plasmid DNA.The plasma DNA solution is not a pure substance and contains some impurities that may have contaminated the solution. These impurities present include RNA’s, cellular proteins and some salts and they would have been added to the solution during the transition from the old isopranol-treat. tubes to the new ones, and some of the white precipitates may have been present in the micropipette. The plasmid DNA gets resuspended in TE so as to remove any remaining RNA’s and it should stay stored in ice, to prevent any degradation of the DNA.
Conclusion
Alkaline lysis is a useful technique for isolating plasmid DNA from the cell without causing any harm to or degradation of the DNA since the many solutions involved in the procedure play distinct functions. The existence of the white precipitate after the neutralizing solution was added, which shows that the genomic DNA did separate from the plasmid DNA and the clear solution indicates the plasmid being isolated from the remainder of the cell, is evidence that the experiment was successful. The contaminants found in the final product are evidence of slight inaccuracies of the experiment
.
References
Andreou L. (2013). Methods in Enzymology (online). (cited 14 March 2023).
Available from https://www.sciencedirect.com/topics/neuroscience/plasmid-dna
Bashkirova E. (2005). Applied mycology and biotechnology (online). (cited 14 March 2023). Available from: https://www.sciencedirect.com/science/article/abs/pii
Guruatma J. (2010, April 12). Alkaline Lysis. ASU - Ask A Biologist. Retrieved 16 March 2023 from https://askabiologist.asu.edu/alkaline-lysis
