Plasmids - Lab Report Example

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To perform plasmid miniprep to isolate pBlueSkript KS II (+) from E.coli and to perform double restriction digestion on pProEx with the enzymes BamHI and Hind III to release the FtsZ insert. …
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Plasmids are extrachromosomal DNA present in the bacterial species. They are double stranded DNA which forms circles with size ranging from 1 kb to 200kb ( kilobase ). Plasmids are very advantageous for the genetic engineering. Plasmids code for many antibiotic regions and they have the ability to accept the gene of interest. The transformation of our gene of interest into the plasmid is called recombination and the bacteria are called recombinant bacteria. Thus plasmids can be used as cloning vehicles or vectors. The first step of transformation is the isolation ofhte plasmid DNA from the given bacteria culture. The basic method or DNA isolation is
1. Cutluring the host cell containing the Plasmid DNA.
2. Harvesting and lysing the cell to separate the DNA from the cell organelles.
3. Separation of chromosomal DNA and Plasmid DNA through precipitation method.
4. Plasmid DNA isolation and purification.
Since both chromosomal and plasmid DNA will remain in the solution, the method to isolate plasmid DNA from the Chromosomal DNA is precipitation method. Larger DNA molecules (i.e. chromosomal DNA), bound to the proteins are separated from the Plasmid DNA when the protein is precipitated. The plasmid DNA which remains in the solution is then precipitated using ethanol.
Method: 1. A single colony of Bacteria containing the pBlueSkript KS II was grown overnight in the Luria Betroth overnight with ampicillin as the antibiotic. 2. From the overnight culture, 1.5 ml of the culture was taken in the centrifuge tube and centrifuged at maximum speed for 1 minute. 3. The supernatant containing the medium is discarded and the cell pellet was kept as dry as possible. 4. The cells were resuspended in the 100µl of GTE buffer and mixed gently using the pipette to ensure that no cell pellets remain in the solution. 5. To the cell pellets, 200 µl of cell lysis buffer was added at room temperature. The tube was mixed gently by inverting the tube up and down five times and incubated at ice for 5 minutes. 6. To the mixture 150 µl neutralization buffer was added and again inverted gently up and down 5-6 times. 7. The mixture was centrifuged at maximum speed for 10 minutes and the supernatant was added to the new tube. 8. To the supernatant, 1000 µl of 100% ethanol was added to precipitate the DNA. 9. The tube is centrifuged for 10 minutes in maximum speed. 10. The supernatant was removed from the tube and to the whitish DNA pellet, 1ml of 70% ethanol was added and the tube was inverted several times and centrifuged at maximum speed for 2 minutes. 11. The supernatant was removed from the solution and to the DNA, 500 µl of 70% ethanol was added as final wash. The tube was again centrifuged at top speed for 2 minutes and the DNA pellet was obtained. 12. The pellet was resuspended in 40 µl of 10mM Tris- HCl with RNase. The tube was mixed by flicking the tube and incubated at 37° C for 5 minutes. 13. 5 µl of the Plasmid DNA was transferred to sterile microfuge tube and was labeled as B3- 5 µl PKS II- southern blot and stored at -20°C. Result and discussion: The DNA was extracted from the culture using the miniprep method. The plasmid DNA obtained in this method is used for the transformation process. Answer 1: Ampicillin is an antibiotic that resists the growth of the ampicillin senstitive strains when added to the medium. As our plasmid PKS II codes for ampicillin gene, ampicillin was induced in the growth medium to avoid contaminants. Answer 2: RNase is the enzyme that cleaves the RNA present in the given sample. RNA are the contaminants seen along with the plasmid DNA. Hence RNase was added to cleave the RNA. Answer 3: We can use alkaline lysis/ phenolic extraction method or alkaline lysis/PEG ...Download file to see next pagesRead More
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