AH109 Yeast Competent Cells

YCC-001

AH109 Yeast Competent Cells

100 μl*50

The AH109 strain is derived from the PJ69-2A yeast strain, and the introduction of the lacZ reporter gene into PJ69-2A gave birth to AH109. The MATa type can be directly transformed into the plasmid or mating with the MATa-type yeast strain Y187 for protein interactions or sieve library assays.

MATa, trp1-901, leu2- 3, 112, ura3-52, his3-200, gal4△, gal80△, LYS2:GAL1_UAS-GAL1_TATA-HIS3, GAL2_UAS-GAL2_TATA-ADE2, URA3:MELI_UAS- MEL1_TATA-lacZ

trp1, leu2

lacZ, HIS3, ADE2, MEL1

PGBKT7, PGADT7

The four reporter genes of AH109 are initiated by three promoters (GAL1, GAL2, MEL1). Only the 17 bp core region recognized by GAL4 is the same among these three promoters. In contrast, the rest of the promoters are different, which greatly reduces the probability of false-positive yeast two-hybrid crosses.

Transformation efficiency >10⁴ cfu/ug DNA as assayed by PGADT7 plasmid.

1). 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.
2). 30 ℃ water bath for 30 min, every 10 min gently turn over and mix 6-8 times.
3). Add 20 μl of dimethyl sulfoxide to each branch (used to improve the conversion efficiency, can not be done).
4). 42 ℃ water bath for 15 min, every 5 min gently turn over and mix 6-8 times.
5).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).
6).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). Competent cells are best melted on ice, 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