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Fluorescent protein vector for directional selection of PCR clones
S Dabrowski1, A Brillowska-Dabrowska, J Kur
1Technical University of Gdańsk, Department of Microbiology, Poland.
Biotechniques
|November 1, 2000
Summary
Enhanced yellow fluorescent protein (EYFP) facilitates directional cloning of blunt-ended PCR products. This reporter system simplifies clone analysis through visual inspection, reducing time and labor.
Area of Science:
- Molecular Biology
- Biotechnology
Background:
- Green fluorescent protein (GFP) is a widely used reporter protein.
- Enhanced yellow fluorescent protein (EYFP) is a variant of GFP.
Purpose of the Study:
- To develop a reporter system for directional cloning of blunt-ended PCR products using EYFP.
- To simplify the analysis of cloned PCR products.
Main Methods:
- Constructed a pUC18-derived plasmid with an EYFP reporter gene.
- Cloned blunt-ended PCR products into the SmaI site of the plasmid.
- Designed reverse PCR primers with a ribosome binding site (rbs) for EYFP expression.
Main Results:
- EYFP expression is dependent on the proper orientation of the rbs at the 3' end of the insert.
- Successful directional cloning yields white fluorescent colonies.
- Analysis can be performed via simple visual inspection with a transilluminator.
Conclusions:
- EYFP serves as an advantageous reporter system for directional cloning of blunt-ended PCR products.
- The method offers a convenient approach to reduce time and labor in clone analysis.