Construction of cDNA libraries

0.0 Source of mRNA (in this case, reticulocytes) l Isolate mRNA. 5' 3’ I Add oligo dT primers. 3'T'I'T'I'T'f'5’ 9—? Synthesize first strand cDNA using reverse transcriptase. 3'M 5' 5'—3’ Partially degrade mRNA using RNase H. 3'—TTTTTS‘ 5'-------3' Synthesize second strand cDNA using DNA polymerase and remaining mRNA fragments as primers. 3'—TTTTTT5‘ S1 nuclease Protect EcoRl sites in cDNA from blunts ends. digestion using EcoRl methylase. 3'—TTTTTTS' 9—? l Ligate linkers containing EcoRl sites. .— EcoRl EcoRl l Digest with EcoRI and clone into vector. (DNA inseq EcoRl , EcoRI Clones of genes expressed at high levels in reticulocytes will appear at greater frequency in this cDNA library than clones of genes expressed at a low level.