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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
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Even though there exist no certainty that a gene recombinant product will be accumulated in the Escherichia coli (E coli) at levels that are high, in biologically active foam, and in full length, a significant magnitude of efforts has been put towards bettering the versatility and performance of the microorganism.
In actual sense, the recipient will have the same combination of genes that are used in the plasmid. this is a type of combining genes from the donor to the recipient, in making a strong production. For instance, during the 1970’s researchers had started the research on how genes could be combined from a donor to the recipient.
This report examined the features of E coli in respect to the expression systems with an emphasis on the limitation number that have been addressed. For a high gene dosage to be achieved, the heterologous cDNAs are normally cloned into replicate plasmids in a fashion that is relaxed and are normally existence at 15-60 copies per cell.
The use of rabies virus as vaccine for HIV and other viral diseases has brought in their efficiency to acts as pseudo titers. The level of expression of the pseudo titers in the HIV -1 is very less using the pl 18 plasmid containing RV277 glycoprotein gene.
The paper gives a review of E-Coli transformed by plasmid DNA using a rapid method and can be categorized into four stages. The first is Pre-incubation stage. In this the cells are immersed in a cat ions solution and incubated at 0ºCThis immersion is supposed to help the negatively charged phosphates of lipids in the E-Coli membrane.
freundii. The biochemical tests also show that fertilizer had the best identification compared to the three samples collected. The biochemical tests were done and used in reference to the information available on the Bergy’s Manual of Determinative
For the first experiment, the plasmids containing the mutant and wild type LHX3 and LHX3b isoforms genes were constructed and then cultured with human embryonic kidney 293T cells. The controls were set up by culturing the cells with empty DNA vector plasmids. The plasmids were taken up by the cells to express the genes encoded by these plasmids.
This research aims to evaluate and present bacterial transformation and gene expression. The research will demonstrate two important genetic engineering tools used for the expression of the foreign gene of interest in the given bacterial cell and three conditions required for the transformation.
EXPRESSION OF RECOMBINANT TICK HISTAMINE-BINDING PROTEIN TC11485 FROM Rhipicephalus microplus USING PPICZ-ALPHA IN Pichia pastoris Abstract Histamine-binding proteins (HBP) are ubiquitous biomolecules with pharmacologic importance, since they have an immunosuppressing activity, which can help prevent adverse health conditions such as histamine shock.
30 Pages(7500 words)Lab Report
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