C dna genomiclibrary3

Subculture colony producing insulin onto new agar plate

Oligo(dT)

primer

Fish pancreas

extract mRNA

AAAnA-3’5’AAAnA-3’5’mRNA

TTTn T-5’

poly(A) tail AAAnA-3’5’

TTTn T-5’3’Reverse transcript

ase

dNTPs

mRNA mRNA

cDNAOligo(dT)

primer

Alkali digestion of mRNA template/RNaseH

TTTnT-5’

3’

TTTn T-5’

AAAnA-3’

dsDNA with a ss hairpin susceptible

to S1 nuclease

cDNA

DNA polymera

se I

dNTPs

S1 nuclease

TTTn T-5’

AAAnA-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

DNA sequence analysisDNA sequence analysis

• The dideoxy nucleotide technique (Sanger)

DNA sequence analysisDNA sequence analysis