Once mRNA is purified, an oligo-dT primer (a short sequence of deoxy-thymidine nucleotides) is bound to the poly-A tail of the RNA. The primer is required to initiate DNA synthesis by the enzyme reverse transcriptase. This results in the creation of RNA-DNA hybrids where a single strand of complementary DNA is bound to a strand of mRNA. To remove the mRNA, the RNAse H enzyme is used to cleave the backbone of the mRNA and generate free 3-OH groups, which is important for the replacement of mRNA with DNA. DNA polymerase I is then added, the cleaved RNA acts as a primer the DNA polymerase I can identify and initiate replacement of RNA nucleotides with those of DNA. This is provided by the sscDNA itself by coiling on itself at the 3 end, generating a hairpin loop. The polymerase extends the 3-OH end, and later the loop at 3 end is opened by the scissoring action of S nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids.
The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.