Materials and Reagents

1. Petri Dishes with Clear Lid (Fisherbrand^TM, catalog number: FB0875712)

2. Toothpicks (flat, sterilized)

3. Slides and coverslips (e.g., Fisherbrand, catalog numbers: 12-550-A3 and 12-542A)

4. Yeast strains

   1. Donor and recipient strains of complementary mating type; MATa and MATα.

   2. One of these, ideally the donor, must contain the kar1-1 mutant allele.

   3. Unique auxotrophic or antibiotic markers that differentiate donor and recipient strains to significantly streamline isolation and confirmation of cytoductants.

   4. To use the plasmiduction protocol described in Section D below, the donor and recipient strains should share at least one auxotrophic marker that will be complemented by the plasmid. Alternatively, a plasmid conferring antibiotic resistance (such as G418 resistance) could be used.

   5. One additional yeast strain (referred to generically throughout this protocol as your “tester strain,” for instance BY4741, BY4742, or another favorite strain; it must meet the following conditions).

      1. Must be of opposite mating type relative to your recipient strain.

      2. Should be wild type for the KAR1 gene.

      3. Should include unique auxotrophic markers relative to your recipient strain to facilitate diploid selection.

      4. Use of this strain to screen cytoductants for fluorescent aggregates (as described in Step B4a) demands two additional requirements. First, the strain must share at least one auxotrophic marker relative to your recipient strain that facilitates maintenance of a plasmid necessary to confirm prion status of your cytoductants (as outlined below in Step B4a). For example, if using a URA3 plasmid, both your recipient and tester strain must be ura3. Second, the strain should be devoid of all prions, but minimally must be devoid of the prion being cytoduced. If prion status of this tester strain is uncertain, cure the strain using guanidine hydrochloride (as described below in Step B4b). Regardless of whether you suspect that the tester strain is free of prions or have cured the strain yourself, it is prudent to verify that the strain contains no prions by visualizing cells via fluorescent microscopy as described (Step B4a, focusing on parts i., and v.-vii. only, as mating will not be required). If properly cured, these cells will have diffuse fluorescence under the conditions described in Step B4a.

         Note: This protocol focuses predominately on Hsp104-dependent prions and thus describes the guanidine hydrochloride method for curing these prions. If you are working with prions that require an alternative curing method, such as [GAR⁺] or [SMAUG⁺], then curing of your tester strain should be completed using your prion specific curing method rather than that described in Step B4b.

5. Plasmids for prion identification or plasmiduction

   1. To detect [PSI⁺] using fluorescence microscopy, plasmid with fluorescently tagged Sup35 protein (e.g., pCUP1-Sup35-GFP, with URA3, Addgene.org #1087)

   2. To detect [PIN⁺] using fluorescence microscopy, plasmid with fluorescently tagged Rnq1 protein (e.g., pCUP1-Rnq1-YFP, with URA3, Addgene.org #15596)

   3. Plasmids that provide optional antibiotic or auxotrophic selection (for the plasmiduction protocol).

6. Media/Agar plates

   1. Dehydrated Agar for solid media (Bacto^TM, catalog number: 214030; 2 kg, or smaller quantity)

   2. YPD (ingredients, recipes in separate section below)

      1. Yeast Extract, low-dusting (LD) (Gibco^TM, Difco^TM, catalog number: 210933)

      2. Peptone (Gibco^TM, Bacto^TM, catalog number: 211677)

      3. Dextrose Glucose, Anhydrous, 2 kg (BD^TM Difco^TM, catalog number: 215510 or smaller quantity)

   3. Components for Synthetic Dropout plates

      1. Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate (Difco^TM, catalog number: 233520)

      2. Ammonium sulfate, 99.5%, for analysis (ACROS Organics^TM, catalog number: 205870010)

      3. Nutrients for dropout powders (for an exhaustive list, including relative proportions, see Sherman, 2002)

         1)

         Amino acids: L-arginine-HCl, L-histidine, L-leucine, L-lysine mono-HCl, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine, L-valine (Sigma-Aldrich)
         2)

         Adenine hemisulfate salt (Sigma-Aldrich, catalog number: A-9126)
         3)

         Uracil (Sigma-Aldrich, catalog number: U-0750)
         4)

         Some yeast strains may have additional unique auxotrophic markers and require corresponding nutrients (Sherman, 2002).

   4. Cycloheximide solution (for detailed protocol B only, see Recipes)

      1. Cycloheximide (Sigma, catalog number: C7698)

      2. DMSO (Sigma, catalog number: D8418)

   5. Ethidium bromide (if recipient strain needs to be rendered rho⁰ for appropriate cytoduction strategy (Sigma, catalog number: E8751)

   6. Glycerol (Sigma, catalog number: G-5516)

   7. Possibly one or more yeast antibiotics dependent upon cytoductant selection strategy. Two common yeast antibiotics are geneticin (Sigma, catalog number: A-1720; Winzeler et al., 1999) and nourseothricin (GoldBio, catalog number: N-500-1; Haarer et al., 2007).

7. Glass beads for spreading yeast cells (Zymo Research, catalog number: S1001)

8. Reagents for yeast transformation (Gietz, 2014)

   1. ssDNA (Sigma, catalog number: D7656)

   2. PEG 3350 (Sigma, catalog number: 88276)

   3. Tris (C₄H₁₁NO₃, GoldBio, catalog number: T-400-1)

   4. EDTA (EMD, catalog number: EX0539-1)

   5. Lithium Acetate (Sigma, catalog number: L4158)

9. Guanidine Hydrochloride (Sigma, catalog number: G4505)

10. Copper Sulfate (Sigma, catalog number: C1297)

11. Media (see Recipes)

12. 10 mg/ml Cycloheximide stock solution (see Recipes)

13. YPD (or YPGlycerol) plates with 10 μg/ml Cycloheximide (see Recipes)

14. 2.5 mg/ml Ethidium Bromide (ETBr; see Recipes)

15. YPD plates with 25 μg/ml EtBr (see Recipes)

16. 5 M Guanidine Hydrochloride (GuHCl; see Recipes)

17. YPD plates with 5 mM GuHCl (see Recipes)

18. SGlycerol-selective plates (see Recipes)

Equipment

1. Fluorescent microscope (to monitor the presence of prion via fluorescent tag)

2. Replica plater/velveteen squares (Fisher Scientific, catalog number: 09-718-1)

3. Autoclave

4. Incubator (30°C)

5. Erlenmeyer Flasks (1 L for media preparation)

   (All equipment is available from any standard scientific supply distributor; specific brands/models are not required)

Procedure