AH109 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. Store it at -20℃ for standby after use. Thaw Carrier DNA on ice before subsequent use. Re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles. Competent cells are best thawed on ice. Transformation with high-concentration plasmid can correspondingly reduce the final bacterial amount for plating. Saccharomyces cerevisiae is temperature-sensitive with an optimal growth temperature of 28℃-30℃; temperatures above 31℃ will affect yeast growth. The growth rate of yeast in defective medium is slower than that in YPDA medium，and the more defective components in the medium，the slower the growth. AH109 is a MATa-type strain of Saccharomyces cerevisiae developed by Clontech. It is a classic strain for GAL4 system yeast two-hybrid assays，derived from PJ69-2A with an additional lacZ reporter gene，and is often mated with the MATα-type Y187 strain for the screening and verification of protein-protein interactions. The transformation markers are trp1 and leu2，and the reporter genes include lacZ，HIS3，ADE2 and MEL1. The AH109-GAL4 yeast two-hybrid system requires two supporting plasmids，pGBKT7 and pGADT7. pGBKT7 carries the TRP1 selection marker and is used to express the fusion protein of DNA-BD and bait protein; pGADT7 contains the LEU selection marker and is applied to express the fusion protein of AD and prey protein. AH109 harbors four reporter genes: lacZ，HIS3，ADE2 and MEL1. The transformation efficiency of AH109 competent cells detected with pGADT7 plasmid (7988 bp，Amp?) is higher than 10? cfu/μg DNA. Genotype: MATa，trp1-901，leu2-3，112，ura3-52，his3-200，gal4Δ，gal80Δ，LYS2::GAL1UAS-GAL1TATA-HIS3，MEL1 GAL2UAS-GAL2TATA-ADE2，URA3::MEL1UAS-MEL1TATA-lacZ。