Autosticky PCR

Directional Cloning of PCR Products with Preformed 5– Overhangs
  • József Gál
  • Miklós Kálmán
Part of the Methods in Molecular Biology™ book series (MIMB, volume 192)

Abstract

The polymerase chain reaction (PCR) is a method of central importance in molecular biology (1,2). The DNA fragment of interest is often amplified for cloning purposes. A frequently used experimental approach is to include extra restriction endonuclease cleavage sites in the amplification primers, digestion of the PCR product with the corresponding enzymes, and ligation of the product to a linearized cloning vector (3). However, the efficiency of cleavage by certain restriction endonucleases is rather low because of the cleavage site(s) being too close to the termini of a DNA fragment (4,5), and internal restriction sites of the fragment might also complicate the cloning.

Keywords

Glycerol Phenol Chloroform Acetonitrile MgCl2 

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • József Gál
    • 1
  • Miklós Kálmán
    • 1
  1. 1.Bay Zoltán Foundation for Applied ResearchInstitute for BiotechnologySzeged

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