Expanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast.
Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins.
Moqtaderi
Zarmik
Z
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Struhl
Kevin
K
D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D015825:Chromosomes, Fungal / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D019474:Transcription Factors, TFIII / Q000235:genetics / Q000378:metabolism; D000098832:Transcription Factor TFIIIC
D000939:Epitopes; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC