Unit

UNIT 2.1 Construction of Small-Insert Libraries from Genomic DNA

  1. Thomas J. Hudson

Published Online: 1 MAY 2001

DOI: 10.1002/0471142905.hg0201s00

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Hudson, T. J. 2001. Construction of Small-Insert Libraries from Genomic DNA. Current Protocols in Human Genetics. 00:2.1:2.1.1–2.1.6.

Author Information

  1. Whitehead Institute, Cambridge, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: FEB 1994

Abstract

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.