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Chapters
- 00:05Title
- 01:30In Silico Design: Final Plasmid and Overlapping Primers
- 03:58Amplification of the DNA Fragments
- 05:44DNA Purification Using Magnetic Beads
- 06:58Assembly Cloning Reaction and Transformation of the Products in E. coli
- 08:21Results: Cloning of CstF-54 and CstF-64 Mutants Under the Transcriptional Regulation of Human Elongation Factor 1α Promoter
- 09:47Conclusion
Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.
