Subcloning Of Fungal CDNA From PBK Lab 
Subcloning of fungal cDNA from pBK-CMW into a plasmid vector pUC19 using fungal gene CIH
Introduction
A plasmid is a circular, double stranded DNA molecule that replicates independently of the chromosome DNA within a cell.pUC19 is one of the most commonly plasmid cloning vector used due to its high copy replication number
(approx. 100 copies per cell), ampR (ampicillin resistance gene) andterminal fragment of β -galactosidase (lac Z). It is circular double stranded and it has
2686 base pair length from which 54 are multiple cloning sites polylinker that contains unique sites for specific restriction endonucleases. pUC19 contains ampicillin resistance gene, which allows selection for bacteria that has received a copy of the vector molecules as recombinants. Therefore the foreign DNA successfully inserted can be easily identified from the nonrecombinants based on the colour difference of colonies on growth media.
Restriction endonucleases are enzymes that can be used to cut DNA into fragments; EcoR1 and Xba1 can be used as they both work optimally in the same reaction buffer and cut in the middle of the recognition sequence resulting in sticky ends which base pair with the complementary sticky ends that accept the fungal gene.
DNA treatment ligase covalently joins the DNA fragments together in order to create a recombinant DNA molecule. (Coventry University, 2014)
The fungal gene CIH-1 isolated from the fungus Colletotichum lindemuthianum that is approximately 600 bp long is inserted into pUC19. The
CIH-1 cDNA is cloned into a vector called pBK-CMV, used for cloning and expressing DNA in mammalian cells; is 4518 bp but can rise up to approximately 5100 bp hence with CIH-1 insertion. (Coventry University,
2014).
The DNA introduced into host bacteria carry a gene fragment that encodes the carboxyl portion of beta-galactosidase and allows its replication driven by
ATP. The Lac Z’ gene contains unique restriction sites, the insertion of DNA
within these
Introduction
A plasmid is a circular, double stranded DNA molecule that replicates independently of the chromosome DNA within a cell.pUC19 is one of the most commonly plasmid cloning vector used due to its high copy replication number
(approx. 100 copies per cell), ampR (ampicillin resistance gene) andterminal fragment of β -galactosidase (lac Z). It is circular double stranded and it has
2686 base pair length from which 54 are multiple cloning sites polylinker that contains unique sites for specific restriction endonucleases. pUC19 contains ampicillin resistance gene, which allows selection for bacteria that has received a copy of the vector molecules as recombinants. Therefore the foreign DNA successfully inserted can be easily identified from the nonrecombinants based on the colour difference of colonies on growth media.
Restriction endonucleases are enzymes that can be used to cut DNA into fragments; EcoR1 and Xba1 can be used as they both work optimally in the same reaction buffer and cut in the middle of the recognition sequence resulting in sticky ends which base pair with the complementary sticky ends that accept the fungal gene.
DNA treatment ligase covalently joins the DNA fragments together in order to create a recombinant DNA molecule. (Coventry University, 2014)
The fungal gene CIH-1 isolated from the fungus Colletotichum lindemuthianum that is approximately 600 bp long is inserted into pUC19. The
CIH-1 cDNA is cloned into a vector called pBK-CMV, used for cloning and expressing DNA in mammalian cells; is 4518 bp but can rise up to approximately 5100 bp hence with CIH-1 insertion. (Coventry University,
2014).
The DNA introduced into host bacteria carry a gene fragment that encodes the carboxyl portion of beta-galactosidase and allows its replication driven by
ATP. The Lac Z’ gene contains unique restriction sites, the insertion of DNA
within these
References: 1. Green E. (2014) 216 BMS Molecular Genetics: DNA Cloning Labs 1-3. 2. Brown, T. A. (2002) Genomes, 2nd ed. Oxford 3 4. Kerbs, J. et al. (2011) Lewin’s Genes X. 11th ed. London 5 7. Thermo Fisher Scientific (2014) Agarose gel electrophoresis tips & tricks [online]Available at https://www.lifetechnologies.com/uk/en/home/life-science/pcr/elevatepcr-research/agarose-content-with-tips-and-tricks.html .[Accessed 10 Dec 2014)