One‐Step Enzymatic Purification of PCR Products for Direct Sequencing

Jae Bum Kim1, Seth Blackshaw1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.6
DOI:  10.1002/0471142905.hg1106s30
Online Posting Date:  November, 2001
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Abstract

The polymerase chain reaction (PCR) has become a powerful and widespread method for analyzing DNA. This protocol offers convenient enzymatic purification of PCR products for direct sequencing without the need for further subcloning, precipitation, or column purification. All reactions may be performed in 96‐well PCR plates and are compatible with large‐scale sequencing. This method is designed to be convenient and rapid. It is suitable for a variety of PCR templates, including plasmid, lambda, and genomic DNA.

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • PCR product to be sequenced (e.g., unit 7.1 or CPMB UNIT ), 25 to 250 ng/µl
  • 1 U/µl shrimp alkaline phosphatase (ShrAP; e.g., USB)
  • 10 U/µl exonuclease I (Exo I; e.g., USB)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 2D)
  • PCR tubes or 96‐well PCR plate
  • Thermal cycler to accommodate tubes or 96‐well plates
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

Literature Cited
   Hanke, M. and Wink, M. 1994. Direct DNA sequencing of PCR‐amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques. 17:858‐860.
   Werle, E., Schneider, C., Renner, M., Volker, M., and Fiehn, W. 1994. Convenient single‐step, one tube purification of PCR products for direct sequencing. Nucl. Acids Res. 22:4354‐4355.
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