Lecture 1 Applied Molecular Biology What does gene cloning entail? 1) Vector cut open with restriction endonuclease (Restriction) 2) Linearized vector and gene ligated together (Ligation) 3) Transform into competent cells (Transformation) 4) Vector replication 5) Select for cells containing vectors over those that don't (Primary Selection) 6) Cell multiplication (Growth and Secondary Selection) We can influence restriction, ligation, transformation, primary selection and secondary selection We can't influence vector replication and cell growth DNA cut at specific sequences with restriction endonuclease. Most vectors are engineered to have just one recognition site for each group of restriction enzymes. Most but not all restriction enzymes recognise palindromes Ligase - reseals the phosphodiester backbone of DNA, joins the 5' phosphate to the 3' OH this is done 4x when inserting gene into vector Transformation - Vector taken up by cells. Uses ice cold CaCl2. This process is very inefficient ffi1 in 1fifififi cells pick up a plasmid. Extremely unlikely that a cell will pick up 2 plasmids. Therefore, every colony gives rise to clones of one single plasmid Vector replication - Multiple copies of the same vector are made inside a cell, up to a predefined copy number encoded on the plasmid Selection/Cell multiplication - only cells that have taken up the plasmid will be able to grow due a selectable marker e.g. ampicillin resistance Secondary selection - Restriction sites are located within a non-essential gene that provides a secondary marker that we can observe e.g. such as restriction sites in the B-galactosidase gene which makes a blue colour. If the plasmid religates to itself without up taking the gene then the colonys will stay blue. If gene taken up it disrupts the B-galactosidase gene and colonys will be white. Distinguishes recombinant from non recombinant.