EGY48 Yeast Competent Cells

YCC-006

EGY48 Yeast Competent Cells

100 μl*50

EGY48 strain is a LexA system yeast two-hybrid strain, MATα type, which can be directly transformed into plasmids to verify protein interactions or screen libraries. The reporter gene UAS ( upstream activation sequence) is derived from LexA op(x6) and initiates LEU2 expression only when Bait and Prey interact. Plasmid pLexA screening flag HIS3 was used to express a fusion protein of DNA-BD with the target protein (Bait). Plasmid pB42AD screening flag TRP1 was used to express a fusion protein of AD with the target protein (Prey). The reporter plasmid p8op-LacZ screening marker is URA3, the reporter gene is LacZ, the reporter gene UAS is derived from LexA op(x6), and LacZ expression is initiated only when Bait and Prey interact.

MATα, ura3, his3, trp1, LexAop (x6)-L

his3, trp1, ura3

LEU2

pLexA, pB42AD, p8op-LacZ

Transformation efficiency >10⁴ cfu/ug DNA as assayed by pGBKT7 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.

Competent cells are best melted on ice, PEG solution will precipitate at low temperatures, please dissolve completely at room temperature before use.

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