Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-3xHA/

Uses: Gene disruption, analysis of gene expression, creating fusion to GFP, HAT epitope-tagging protein at range of sites, creating conditional alleles.

In more detail: mTn-3xHA/GFP can be used easily to create a library of insertions, each at a different site in a given gene. The mutagenized DNA is then transformed into yeast, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. Insertions that generate in-frame fusion of the coding region to GFP can be used to monitor and quantify gene expression, via assays for fluorescence activity. Localization of the GFP fusion protein can be examined by fluorescence microscopy. The transposon can also be excized by Cre-mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus a 274-bp insertion containing sequences encoding the 3xHA tag and the factor Xa protease cleavage recognition site. When GFP is fused in-frame to the gene of interest, the excision event results in an in-frame insertion of 93 amino acids, called a HAT tag, into the encoded protein. The HAT tag allows immunodetection of the protein. Insertion of the HAT tag also has the potential to create conditionally-defective forms of the protein.

The accession number for mTn-3xHA/GFP is U54830.

A kit for mutagenesis of a yeast gene with mTn-3xHA/GFP is available.

• See what the kit contains
• Order the kit

Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-3xHA/GFP

Please read this whole document before you start!

Shuttle mutagenesis

1. Clone target gene into vector pHSS6.
   • pHSS6 is in strain R1123; map given below.
   • Delete as much of the polylinker as possible as sometimes transposon 'hot-spots' into it.
   • Select transformants on LB Kan40.
2. Transform target plasmid into competent cells of R1236/B211.
   • Select on LB Kan40 Cm34.
3. Transfer F::mTn-3xHA/lacZ into cells by mating with strain #111/B428.
   • Grow strains overnight with antibiotic selection (Tet3 for #111/B428).
   • Subculture 1:100 in fresh LB medium (no antibiotics). Grow at 37^oC to early log phase (when cell swirls are visible). The recipient strain (R1236/B211) can be denser than the donor (#111/B428).
   • Mix 200 ul of each strain. Incubate at 37^oC without agitation for 20 min to 1 hr.
   • Plate as 100 ul aliquots onto LB Tet3 Kan40 Cm34. Do the Control: Spot the starting strains onto this media.
   • Grow 1-2 days at 30^oC. Now you have cointegrates.
   • Set up strain #70/B425 in Sm50 Cm34 overnight.
4. Resolve cointegrates by mating to strain #70/B425.
   • Elute colonies from plates: put 2 mls of LB on the plate, scrape off the colonies with a speader. This is your eluate. You should have several thousand colonies at least.
   • Dilute overnight culture of strain #70/B425 1:100 without antibiotic. Dilute eluate to roughly same density. Grow and mate as before.
   • After mating for 20 min to 1 hr, plate 100 ul aliquots on LB Tet3 Kan40 Sm50 and grow overnight at 37
     

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