McClean: GEV Strain Construction
This protocol explains how to insert the GAL1 promoter in front of a gene of interest in the appropriate strain with the GEV machinery (yMM1101 MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2 or similar) so that production of the transcript (and therefore the protein) is inducible with β-estradiol.
Primarily this protocol deals with how to appropriately design primers to insert the GAL1 promoter. Other parts of the protocol (PCR, transformation) are standard and you are referred to other McClean lab protocols for those bits.
This protocol has been modified from Scott McIsaac's (Botstein Lab) protocol. Use of this protocol and the subsequent strains should cite his paper (see references below).
- yMM1101 (MAT a HAP1+ (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2) or a similar strain with the GEV machinery driven by a constitutive promoter. Please note that this strain is DBY12020 from McIsaac, et al 2011.
- yMM1101 (MAT a gal10::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (BY4742)) genomic DNA (see Notes)
- Primers (see below for how to design)
- PCR supplies (see: Takara PrimeStar PCR)
- Transformation supplies (see: Yeast Transformation (S. cerevisiae))
Appearance of Tetrads
Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.
How to distinguish a tetrad from two budded cells adjacent to each other?
- Generally, the spores of a tetrad are smaller than a budded cell.
- Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
- The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Megan N McClean 17:48, 29 June 2012 (EDT) I am incredibly lazy and use the Bust n' Grab protocol for genomic DNA prep. To be even lazier, I never measure the concentration of the genomic DNA that comes out of the protocol, I just dilute it 1:100 with water and use 1μL of that in a 50μL PCR reaction (using Takara PrimeStar polymerase). This almost always works. When it doesn't work it is usually the concentration of the genomic DNA that is the problem, so I just go back and try 1:10 and 1:1000 dilutions. Feel free to not be this lazy, but this is how I do it and it usually works.
- McIsaac, RS et al (2011) Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae Molecular Biology of the Cell 22 4447-4459
- Megan N McClean 14:01, 2 July 2012 (EDT)
or instead, discuss this protocol.