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Vaccine Design, Search for the SARS CoV-2 genome on a genomic library. NML…
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Search for the SARS CoV-2 genome on a genomic library. NML-NCBI is selected as it already has the complete genome of the virus isolated. This will enable us to verify the DNA obtained from the PCR in our lab.
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Since we are dealing with an RNA template, we should perform a RT-PCR. Using reverse transcriptase we would turn RNA into cDNA. Then the PCR performed to amplify our cDNA sequence.
Oligonucleotide primers need to be specific for our gene of interest. The primers are going to hybridize to specific DNA or RNA sequence.
We are going to cut the SARS CoV-2 gene with EcoR and then do the same with our plasmid. By cutting with the same restriction enzyme we will produce sticky ends that are complimentary to each other.
DNA ligase is used to join the "sticky ends" in SARS Cov-2 gene and the ones on our plasmid. Once joined by ligase, our plasmid becomes a recombinant plasmid.
Ecoli takes a plasmid and "hosts" it. Bacteria grows in medium, allowing our gene of interest to ploriferate.
To check that our plasmid has the right gene in it, we will use electrophoresis to separate the fragments of DNA. Once that have been separated, we can examine the gel by examining our bands with the reference controls
After the recombinant DNA is inserted into the host cell, we need to identify the cells within the host that have the presence of our recombinant DNA. We do this by adding an antibiotic to our medium, our recombinant DNA will have resistance to this antibiotic. This enables that the only cells to survive and produce colonies are the recombinant ones, since the non-recombinant ones have no resistance to our antibiotic and die.