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.
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)
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 McClean: Genomic DNA Prep (Bust 'n' Grab Protocol) Bust n' Grab "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 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, 20 July 2011 (EDT)
or instead, discuss this protocol.