Construction of small-insert libraries from genomic DNA

Construction of small-insert genomic libraries involves ligation of a size-selected insert to vector DNA. In this unit, vector DNA is prepared by digestion with a restriction endonuclease followed by phosphatase treatment to dephosphorylate the vector ends. Insert DNA is prepared by complete digestion of DNA with a compatible 4-base-cutting restriction endonuclease (e.g., AluI, HaeIII, RsaI, or Sau3A), as such frequently cutting endonucleases give the highest fragment number in the desired range. Alternatively, fragments may be generated by sonication of genomic DNA. Once the fragments have been generated, they are size selected on an agarose gel. The portion of the gel containing DNA of the desired size is excised and the DNA released from the agarose by digestion with b-agarase. Insert and vector DNAs are then ligated with T4 DNA ligase and used to transform competent Escherichia coli cells to create a small-insert library.