every 50 bp genes are constructed to be approximately 100 bp in length this reduces the number of colonies that must be screened in order to find one containing the proper dna sequence the location at which one gene ends and the next begins is selected based on the natural occurrence of unique restriction enzyme cut sites within the gene resulting in fragments or oligonucleotides of variable length the process is greatly assisted by the use of software which identifies the location and frequency of restriction enzyme sites within a given dna sequence once the gene fragments have been successfully designed common restriction enzyme sites are included on the ends of each fragment to allow ligation of each fragment into a cloning plasmid for example adding tcori and hindiii sites to each gene fragment allows it to be inserted into the polylinker cloning region of puc 19 yanish perron c et al 1985 gene 33 103 119 the 3 u2032 and 5 u2032 single strands of each gene fragment are then synthesized using standard solid phase synthesis with the proper sticky ends for insertion into the cloning vector following cleavage and desalting the single stranded fragments are then purified by page and annealed after phosphorylation the annealed fragments are ligated into a cloning vector such as puc 19 following ligation the plasmids are transformed into dh5 f u2032 competent cells and plated on isopropyl d thiogalactopyranoside iptg 5 bromo 4 chloro 3 indolyl d galactopyranoside x gal plates to screen for the insertion of the gene fragments the resulting colonies which contain gene.