Y1HGold Chemically Competent Cell

For the first use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 min，then place it on ice immediately and store it at -20℃ after use for standby. Thaw Carrier DNA on ice before subsequent use，and re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles. Competent cells are best thawed on ice. The use of high-concentration plasmids for transformation can correspondingly reduce the amount of bacterial solution used for final plating. Saccharomyces cerevisiae is temperature-sensitive with an optimal growth temperature of 28℃-30℃，and temperatures above 31℃ will affect yeast growth. Yeast grows slower in dropout medium than in YPDA medium，and the more deficient components in the medium，the slower the yeast grows. Y1HGold is a MATα strain applied for the GAL4-AbA yeast one-hybrid system，which can be directly transformed with plasmids for library screening assays. Its transformation markers are ura3 and leu2，and the reporter gene is AbAr. The Y1HGold-GAL4-AbA yeast one-hybrid system requires two compatible plasmids: pAbAi and pGADT7. The pAbAi plasmid carries the URA selection marker and is used to construct the pBait-AbAi construct，in which 1 to 3 tandem repeated bait DNA sequences are cloned into pAbAi; the pGADT7 plasmid has the LEU selection marker and expresses the fusion protein of AD (GAL4 C-terminal 768~881 amino acids) and target prey protein. Principle of the GAL4-AbA yeast one-hybrid system: Aureobasidin A (AbA) is a cyclic lipopeptide antibiotic that exhibits toxicity to yeast at a low concentration of 0. 1–0. 2 μg/ml. In yeast strains integrated with the pBait-AbAi construct in the genome，when the prey protein binds to the bait DNA sequence，GAL4 AD activates the expression of AbAr，enabling the strain to grow on medium containing AbA antibiotic. Compared with auxotrophic reporter genes，AbAr features lower background，which can reduce the probability of false positives in yeast one-hybrid assays. The transformation efficiency of Y1HGold competent cells tested with pGADT7 plasmid (7988 bp，Amp?) is over 10? cfu/μg DNA. Genotype: MATα，ura3-52，his3-200，ade2-101，trp1-901，leu2-3，112，gal4Δ，gal80Δ，me4-，MEL1