Journal
BIOTECHNIQUES
Volume 64, Issue 5, Pages 219-223Publisher
FUTURE SCI LTD
DOI: 10.2144/btn-2018-0007
Keywords
In-Fusion; site-directed mutagenesis; vector construction; yeast two-hybrid
Funding
- Startup Fund for Advanced Talents of Zhoukou Normal university [ZKNU2014113, ZKNU2014101]
- Foundation of He'nan Educational Committee [17B180008, 16A180056, 16A180057]
- Natural Science Foundation of He'nan province [162300410346, 162300410107]
- Foundation of He'nan Science and Technology Committee [172102410060, 182102110289, 182102110067]
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Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is r-estriction endonuclease-and ligase-independent, and it is fast and easily performed.
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