Gene Cloning
Cloning of plasmid pUC19 in E.coli bacteria
Introduction
One aspect of the DNA cloning experiments that is carefully considered is the selection of cloning vectors. A variety of vectors have been created, each being suitable for a particular use. One common vector used in laboratories is a plasmid called pUC19. It is 2686 base pairs long and possesses an origin of replication which allows the production of over 100 copies in a competent E.coli cell. It possesses a multiple cloning site (MCS) which is artificially implanted by adding a polylinker sequence to it. The pUC19 plasmid is also altered by inserting a gene that codes for beta-lactamase which confers resistance to the antibiotic ampicillin (Read and Strachan 2011). The MCS occupies the 5’ end of the gene lacZ (Sherwood, Willey and Woolverton 2012). This gene codes for only the alpha-peptide of beta-galactosidase, an enzyme used to break down the disaccharide lactose into glucose and galactose (Read and Strachan 2011). The aim of this experiment is to incorporate a cDNA called CIH-1, from plasmid pBK-CMV, into pUC19.
DNA cloning is dependent on type 2 restriction endonuclease enzymes. They function by cleaving both strands of DNA on specific points known as restriction sites. These sites are reliant on the sequences of DNA that are recognised by them. Different bacterial strains yield varying restriction endonucleases. There are currently over 250 recognition sequences identified (Read and Strachan 2011). Restriction endonucleases can cleave DNA sequences on vectors making them competent for the binding of other DNA fragments cut by the same enzyme. They are thus important tools in the production of recombinant DNA (Ahmed, Glencross and Wang 2011). The first objective of this experiment was to use two restriction endonucleases, EcoR1 and Xba1, to cut pUC19 and pBK-CMV. To ensure that the plasmids were successfully cut, analysis of the plasmid was carried out using gel electrophoresis. Gel
Introduction
One aspect of the DNA cloning experiments that is carefully considered is the selection of cloning vectors. A variety of vectors have been created, each being suitable for a particular use. One common vector used in laboratories is a plasmid called pUC19. It is 2686 base pairs long and possesses an origin of replication which allows the production of over 100 copies in a competent E.coli cell. It possesses a multiple cloning site (MCS) which is artificially implanted by adding a polylinker sequence to it. The pUC19 plasmid is also altered by inserting a gene that codes for beta-lactamase which confers resistance to the antibiotic ampicillin (Read and Strachan 2011). The MCS occupies the 5’ end of the gene lacZ (Sherwood, Willey and Woolverton 2012). This gene codes for only the alpha-peptide of beta-galactosidase, an enzyme used to break down the disaccharide lactose into glucose and galactose (Read and Strachan 2011). The aim of this experiment is to incorporate a cDNA called CIH-1, from plasmid pBK-CMV, into pUC19.
DNA cloning is dependent on type 2 restriction endonuclease enzymes. They function by cleaving both strands of DNA on specific points known as restriction sites. These sites are reliant on the sequences of DNA that are recognised by them. Different bacterial strains yield varying restriction endonucleases. There are currently over 250 recognition sequences identified (Read and Strachan 2011). Restriction endonucleases can cleave DNA sequences on vectors making them competent for the binding of other DNA fragments cut by the same enzyme. They are thus important tools in the production of recombinant DNA (Ahmed, Glencross and Wang 2011). The first objective of this experiment was to use two restriction endonucleases, EcoR1 and Xba1, to cut pUC19 and pBK-CMV. To ensure that the plasmids were successfully cut, analysis of the plasmid was carried out using gel electrophoresis. Gel