The document describes the process of cloning the gene for insulin from fish pancreas mRNA. Key steps include reverse transcribing the mRNA to cDNA using an oligo(dT) primer, digesting the mRNA template, ligating the cDNA into a plasmid vector, transforming E. coli bacteria with the vector, screening colonies for insulin production using radio-labeled antibodies, and sequencing the insulin gene from a positive colony.

3. Subculture colony producing insulin onto new agar plate Oligo(dT) primer Fish pancreas extract mRNA AAA n A-3’ 5’ AAA n A-3’ 5’ mRNA TTT n T-5’ poly(A) tail AAA n A-3’ 5’ TTT n T-5’ 3’ Reverse transcriptase dNTPs mRNA mRNA cDNA Oligo(dT) primer Alkali digestion of mRNA template/RNaseH TTT n T-5’ 3’ TTT n T-5’ AAA n A-3’ dsDNA with a ss hairpin susceptible to S1 nuclease cDNA DNA polymerase I dNTPs S1 nuclease TTT n T-5’ AAA n A-3’ dsDNA Clone fragments onto plasmid vector Recombinant plasmid dsDNA Plasmid Transform E. coli Screen colony producing insulin by replica plating on nitrocellulose membrane followed by staining using radio-labelled antibodies specific to insulin Bacterial colonies on agar plate Colony producing insulin Harvest insulin

10. DNA sequence analysis