Strains (available through the Arabidopsis Biological Resource Center, https://abrc.osu.edu/)
• Y8800, prey strain
• CRY8930, bait strain
CrY2H-seq plasmids (available through the Arabidopsis Biological Resource Center, https://abrc.osu.edu/)
• pADlox prey vector
• pDBlox bait vector
Generating CrY2H-seq bait and prey clones
Entry clones to serve as your genes of interest
LR clonase (Life Technologies)
DH5α-T1R chemically competent E. coli cells (Life Technologies)
SOC powder (MP Biomedicals)
Terrific Broth, granulated (Fisher)
Lithium Acetate dihydrate (Sigma)
Tris Base Ultrapure (US Biological)
EDTA Disodium Salt Dihydrate (US Biological)
Poly(ethylene glycol) 3350 (Sigma)
Salmon sperm DNA 10mg/mL (Sigma)
Dextrose (D-Glucose) anhydrous (Fisher)
Bacto Peptone (BD)
Bacto Yeast Extract (BD)
Drop-out mix synthetic minus leucine, histidine, tryptophan, adenine without yeast nitrogen base (US Biological)
Yeast nitrogen base without amino acids (Amresco)
Adenine hemisulfate salt (Sigma)
L-Histidine monohydrochloride monohydrate (Sigma)
Hygromycin B (Bioworld)
3-Amino-1,2,4-triazole (3-AT; Sigma)
Sodium phosphate monobasic (Fisher)
Sodium phosphate dibasic (Fisher)
Zymolyase 20T (US Biological)
Phusion HF DNA Polymerase (NEB)
Deoxynucleotides Set (BioPioneer)
DNA and sequencing library preparation
SeraMag Speedbeads (GE; 2% v./v. SeraMag Speedbeads, 18% w./v. PEG-8000, 1 M NaCl, 10 mM Tris HCl, 1mM EDTA)
QIAprep 96 turbo kit (Qiagen)
QIAquick gel extraction kit (Qiagen)
QIAquick PCR purification kit (Qiagen)
Illumina sequencing library preparation
End-It DNA End-Repair Kit (Epicentre-Illumina)
Klenow Fragment 3’-->5’ exo- (NEB)
TruSeq DNA Sample Prep Kit (Illumina)
T4 DNA ligase (NEB)
Terrific Broth + antibiotic Dissolve 47.6g to 1L of purified water. Then add 4mL glycerol to mixture and stir to dissolve completely. Autoclave for 15 minutes. Allow to cool to 50 °C and add 1 mL 1000x carbenicillin stock solution per liter terrific broth.
Carbenicillin (1,000x) stock Dissolve 50 mg carbenicillin disodium salt in a total volume of 1 mL deionized distilled water. Filter-sterilize and aliquot. Use at a working concentration of 50 mg/L. Store at −20 °C.
SOC Dissolve 31 g/L of purified water. Autoclave for 15 minutes.
0.5 M EDTA (pH 8.0) Dissolve 93.05 g EDTA (disodium salt, Dihydrate) in 400 mL deionized distilled water. Adjust pH to 8.0 using NaOH and bring the final volume up to 500 mL with deionized distilled water and autoclave.
1 M Tris (pH 7.5) Dissolve 121.14 g Tris base in 800 mL deionized distilled water. Adjust the pH to 7.5 using concentrated HCl and bring the volume up to 1 liter with deionized distilled water and autoclave.
10x Tris-EDTA (TE) buffer (pH 7.5) Mix 100 mL 1 M Tris (pH 7.5), 20 mL 0.5 M EDTA (pH 8.0) and 880 mL deionized distilled water. Autoclave.
50% Glycerol solution Dissolve 500 mL glycerol in a total of 1 L deionized distilled water and autoclave.
1 M Lithium Acetate Dissolve 10.2 g lithium acetate dihydrate in a total volume of 100 mL deionized distilled water and autoclave. Store at room temperature.
50% PEG solution (wt/vol) Dissolve 50 g PEG-3350 in a total volume of 100 mL deionized distilled water and autoclave.
Lithium Acetate/Tris EDTA solution Combine 1 mL 1 M lithium acetate solution, 1 mL 10x TE buffer (pH 7.5), and 8 mL sterile water.
Lithium Acetate/Tris EDTA/PEG solution Combine 1 mL 1 M lithium acetate solution, 1 mL 10x TE buffer (pH 7.5), and 8 mL of 50% PEG solution.
40% Glucose solution Dissolve 400 g D-glucose in up to 1 L deionized distilled water and autoclave.
40 mM Tryptophan solution Dissolve 817 mg of tryptophan in 100 mL deionized distilled water. Filter sterilize, protect from light, and store at 4 °C.
100 mM Histidine solution Dissolve 1.55 g histidine in 100 mL deionized distilled water. Filter sterilize, protect from light, and store at room temperature.
100 mM Leucine solution Dissolve 1.31 g leucine in 100 mL deionized distilled water. Filter sterilize or autoclave and store at room temperature.
1 M 3-AT solution Dissolve 8.4 g 3-AT in a total volume of 100 mL deionized distilled water. Filter-sterilize and aliquot as required. Store at −20 °C for up to 6 months.
YEPD media (liquid and solid) Dissolve 10 g yeast extract, 20 g peptone, and 200 mg adenine hemisulfate in a total of 950 mL deionized distilled water. Adjust the pH to 6.0 with HCl. If preparing solid medium, add 20 g agar (omit if preparing liquid) and autoclave. Add 50mL 40% glucose solution after autoclaving.
Synthetic complete dropout media (liquid and solid) Dissolve 6.7 g yeast nitrogen base, 1.47 g of dropout mix, and 200 mg adenine hemisulfate in 900 mL deionized distilled water. Adjust pH to 5.9 with sodium hydroxide. Add 20 g of agar for solid medium (omit if preparing liquid) and autoclave. Allow to cool to 50 °C, then add 50 mL 40% glucose solution, and 8 mL of each amino acid solution desired. If preparing 1 mM 3-AT medium, add 1 mL of 1 M 3-AT solution.
0.1 M Sodium phosphate buffer pH 7.4 Add 3.1 g of sodium phosphate monobasic and 10.9 g of sodium phosphate dibasic to up to 1 L deionized distilled water. The final pH solution will be 7.4.
Yeast lysis buffer Combine 0.1 M sodium phosphate buffer pH 7.4 with 1% betamercaptoethanol, 2.5mg/mL Zymolyase 20T and 100 ug/mL RNase A. Aliquot and store at -20 °C or make fresh.