Recombinant Dna Technology : Sub Cloning Of Cdna Molecule Cih
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Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
The aim of this experiment was to isolate cDNA molecule CIH-1 (Colletotrichum lindemuthianum CIH1 gene) that is contained in vector pBK-CMV and transfer it into cloning vector pUC19. This was attempted by conducting a restriction digest of vectors pUC19 and pBk-CMV containing CIH-1, using restriction endonucleases Xba1 and EcoR1 and the characterization of recombinant plasmids.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
The restriction digest was performed using restricti...
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...but that it is too small to be seen.
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.