McClean: Golden Gate- Making a multi-part plasmid

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Contents

Overview

This protocol describes how to assmble multiple part plasmids via BsaI digestion and T7 Ligase ligation. It is intended to supplement Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015), as well as its supplementary documents.


Materials

  • BsaI restriction enzyme.
  • T4 Ligase Buffer
  • T7 Ligase
  • dATP
  • DNA to insert


Protocol

  • Make a 50 μL solution of BsaI, T4 Ligase Buffer, 20 fMoles of each DNA type, and DI water.
  • Digest for 2 hours at 37°C. This pre-digest is recommended because BsaI is not fully functional in T4 Ligase buffer. If we could, we would add CutSmart buffer which is ideal for BsaI, but we need T4 ligase buffer to make the ligation step easier.
  • Bring the solution to an additional 0.5mM ATP and add T7 DNA ligase. Note that T4 Ligase buffer already included ATP, but this protocol assumes that half of it degraded over 2 hours at 37°C. Thermocycle according to the following routine:
    • 1 hr at 25°C (give time to ligate)
    • The following 25 times
      • 5 minutes at 37°C (re-digest)
      • 15 minutes at 16°C (re-ligate)
    • 2 hr at 37°C (fully digest)
    • 20 min 80°C (heat-kill)
  • Transform this product into competent e. Coli, and select white colonies on Kanamycin.

Notes

This protocol is more involved than the one recommended in the Yeast Toolkit paper. It was designed to improve the efficiency of the transformation, and the extra steps are much better than the possibility of redoing the entire experiment. The paper's instructions were designed for simplicity and speed. These instructions are designed to make it work.

References

Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015)

Contact

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