Y1HGOLD Yeast Competent Cells

YCC-026

Y1HGOLD Yeast Competent Cells

100 μl*50

Y1HGold is a GAL4-AbA yeast one-hybrid system strain, MATα type, which can be directly transformed into plasmids for sieve library assay. Plasmid pAbAi with screening marker URA was used to express the pBait-AbAi construct (1-3 bait DNA sequences repeated in tandem and cloned into pAbAi). Plasmid pGADT7 was screened as LEU for expression of a fusion protein of AD (amino acids 768-881 at the C-terminal position of GAL4) and the target protein (Prey).

MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, gal80Δ, met–, MEL

ura3, leu2

AbAr

pAbAi and PGADT7

AbAr has the advantage of a lower background compared to nutrient-deficient reporter genes and reduces the probability of yeast single heterozygous false positives occurring.

Transformation efficiency >10⁴ cfu/ug DNA as assayed by pGADT7 plasmid.

1). Take 5 µg of pBait-AbAi plasmid, BstBI, or BbsI digestion for 1 h and recover.
2). Take a sterile 1.5 ml EP tube, sequentially add 1-3 μg of pre-cooled target plasmid, 10 μl of Carrier DNA (95-100 ℃ for 5 min in a rapid ice bath, repeat once), and 100 μl of competent cells melted on ice, 500 μl of PEG/LiAc, and gently turn and mix 6-8 times.
3). 30 ℃ water bath for 30 min, every 10 min gently turn over and mix 6-8 times.
4). Add 20 μl of dimethyl sulfoxide to each branch (used to improve the conversion efficiency, can not be done).
5). 42 ℃ water bath for 15 min, every 5 min gently turn over and mix 6-8 times.
6).12,000 rpm instantaneous centrifugation and discard the supernatant, add 1 ml of YPD Plus to each cartridge, and resuscitate at 30℃ for 1 h (used to improve the transformation efficiency, can not be done).
7).12,000 rpm instantaneous centrifugation and discard the supernatant, resuspend with 100 μl of 0.9% NaCl, coat the plate, and incubate at 30℃ for 48-96 hours.

1). PEG solution will precipitate at low temperatures, please dissolve completely at room temperature before use.
2). Transforming a high concentration of plasmid can reduce the amount of bacteria used for plate coating.
3). When transforming 2-3 plasmids at the same time, the amount of plasmid can be increased.
4). The yeast strain is sensitive to high temperatures, and the optimal growth temperature is 27-30℃. Above 31°C, the growth rate and transformation efficiency decreased exponentially.
5). Colonies turning pink is not contamination and is a common phenomenon in yeast cell growth. When the cells are cultured in plates for a few days, the Adenine on the plates is consumed by the yeast and the yeast attempts to synthesize Adenine for utilization through its metabolic pathway. However, in some strains, the ADE2 gene is disrupted and the Adenine synthesis pathway is blocked, and because their ADE4, 5, 6, 7, and 8 genes are normal, the intermediate product AIR accumulates in the cells and causes the colonies to turn pink.
6). Yeast growth in the defective medium was slower than in the YPDA medium, and the more defective components in the medium, the slower the growth. Take the transformation coated plate for example, coated YPDA plate at 29 ℃ culture 48h visible diameter of 1 mm clones; coated SD single-deficient plate 29 ℃ culture 48- 60h visible diameter of 1 mm clones; coated SD double-deficient plate 29 ℃ culture 60- 80h visible diameter of 1 mm clones; coated SD triple-deficient or quadruple-deficient plate 29 ℃ culture 80-90h visible diameter of 1 mm clones.

5 μg/μl, 100 μl

5 ml

Competent cells are transported on dry ice and stored at -80°C. Carrier DNA needs to be stored at -20°C. PEG/LiAc needs to be stored at 4°C.

6 months