Question

How can a cDNA library be prepared?

Answer 1

1. Isolation of mRNA: Extract mRNA from the cells whose genes you want to clone. mRNA serves as the template because it represents expressed genes.
2. Synthesis of cDNA: mRNA is reverse transcribed into complementary DNA (cDNA) using the enzyme reverse transcriptase. This cDNA corresponds only to expressed genes.
3. Conversion to double-stranded cDNA: The single-stranded cDNA is then converted into double-stranded cDNA by DNA polymerase.
4. Insertion into vectors: The double-stranded cDNA is then inserted into cloning vectors (like plasmids).
5. Transformation: The recombinant vectors are introduced into host cells (usually bacteria), creating a cDNA library.
6. Screening: The clones can then be screened to identify and isolate specific genes of interest.