Subcloning of DNA Fragments

Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.16
DOI:  10.1002/0471142727.mb0316s13
Online Posting Date:  May, 2001
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Abstract

The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNAsynthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

     
 
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Table of Contents

  • Construction of hybrid DNA Molecules
  • Alternate Protocol 1: Ligation of DNA Fragments in Gel Slices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Calf intestine phosphatase (CIP) and buffer (optional; unit 3.10)
  • dNTP mix (0.5 mM each; unit 3.4)
  • Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase (optional; unit 3.5)
  • Oligonucleotide linkers (optional)
  • 10 mM ATP
  • 0.2 mM dithiothreitol (DTT)
  • T4 DNA ligase (measured in cohesive‐end units; unit 3.14)
  • recipe2× T4 DNA ligase buffer
  • Additional reagents and equipment for restriction endonuclease digestions (unit 3.1), transformation of E. coli cells (unit 1.8), DNA minipreps (unit 1.6), and agarose or polyacrylamide gel electrophoresis (unit 2.5 or ).

Alternate Protocol 1: Ligation of DNA Fragments in Gel Slices

  Additional Materials
  • Low gelling/melting temperature agarose (SeaPlaque, FMC Marine Colloids)
  • TAE buffer ( appendix 22)
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Figures

Videos

Literature Cited

Literature Cited
   Dugaiczyk, A., Boyer, H.W., and Goodman, H.M. 1975. Ligation of EcoRI endonuclease‐generated DNA fragments into linear and circular structures. J. Mol. Biol. 96:174‐184.
   Struhl, K. 1985. A rapid method for creating recombinant DNA molecules. Biotechniques 3:452‐453.
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