Skip to main content

A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the “Coffee Break Ligation” technique

Abstract

The ligation reaction is widely used in molecular biology. There are several kits available that complete the ligation reaction very rapidly but they are rather expensive. In this study, we successfully modified the ligation buffer with much lower cost than existing kits. The ligation reaction can be completed in 10 min using very low activities such as 0.01 U T4 DNA ligase, and costs only $1 for 100 reactions of 20 μl scale. We name this ligation system the “Coffee Break Ligation” system; one can complete ligation reaction while drinking a cup of coffee, and perform 100 reactions by spending money equivalent to a cup of coffee.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Hayashi K, Nakazawa M, Ishizaki Y, Obayashi A (1985a) Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol. Nucleic Acids Res 13:3261–3271

    PubMed  Article  CAS  Google Scholar 

  • Hayashi K, Nakazawa M, Ishizaki Y, Hiraoka N, Obayashi A (1985b) Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions. Nucleic Acids Res 13:7979–7992

    PubMed  Article  CAS  Google Scholar 

  • Hayashi K, Nakazawa M, Ishizaki Y, Hiraoka N, Obayashi A (1986) Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol. Nucleic Acids Res 14:7617–7631

    PubMed  Article  CAS  Google Scholar 

  • Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28

    PubMed  Article  CAS  Google Scholar 

  • Pheiffer BH, Zimmerman SB (1983) Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res 11:7853–7871

    PubMed  Article  CAS  Google Scholar 

  • Rusche JR, Howard-Flanders P (1985) Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase. Nucleic Acids Res 13:1997–2008

    PubMed  Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Plasmids and Their Usefulness in Molecular Cloning. In: Sambrook J, Russell DW (eds) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, pp 1.1–1.170

    Google Scholar 

  • Zimmerman SB, Pheiffer BH (1983) Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli. Proc Natl Acad Sci USA 80:5852–5856

    PubMed  Article  CAS  Google Scholar 

  • Zimmerman SB, Harrison B (1985) Macromolecular crowding accelerates the cohesion of DNA fragments with complementary termini. Nucleic Acids Res 13:2241–2249

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. B. L. S. Pierce (the University of Maryland University College, 3501 University Blvd. East Adelphi, MD 20783, USA) for editorial work in the preparation of this manuscript. This work was supported in part by Grants-in-Aid and the 21st Century COE Program Special Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Akira Horii.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yoshino, Y., Ishida, M. & Horii, A. A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the “Coffee Break Ligation” technique. Biotechnol Lett 29, 1557–1560 (2007). https://doi.org/10.1007/s10529-007-9429-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-007-9429-z

Keywords

  • Ligation
  • Polyethylene glycol
  • T4 DNA ligase