Frozen ez yeast transformation iitm kit

The coding sequences of AtTFL1, RoFT, RoFTmu1/2/3/4/5, FaTFL1, RoTFL1, and PhFT (all containing the EcoR1 and Sal1 restriction sites at the 5′ and 3′ ends, respectively) were cloned into bait plasmid PGBKT7. Arabidopsis FT (AtFT) was also introduced to the PGBKT7 plasmid, using the Nde1 and Sal1 restriction sites, as a positive control. The full-length Arabidopsis FD coding sequence (AtFD) was cloned into prey plasmid PGADT7 using the Nde1 and BamH1 restriction sites. Yeast cells were transformed using Frozen-EZ Yeast Transformation II^TM kit (ZYMO RESEARCH, USA). Co-transformed yeast cells were selected on SD-Leu/-Trp plates. Interactions were tested on SD-Leu/-Trp/-His/-Ade/X-a-Gal selective media. Three independent clones for each transformation were tested.

pGBKT‐BD bait and pGADT7‐AD prey vectors (Clontech Laboratories) containing cDNA encoding the proteins of interest were cotransformed into the Y2H Gold yeast strain (Clontech Laboratories) using the Frozen‐EZ Yeast Transformation II^TM kit (Zymo Research) following the manufacturer's instructions. Transfected cells were spotted on selective plates of synthetically defined (SD) medium lacking the amino acids tryptophan (Trp), leucine (Leu), histidine, and adenine containing 120 ng/mL aureobasidin A and incubated at 30°C for 3–4 days to verify interaction. Successful transformation of the two plasmids was confirmed by growth on SD/‐Trp/‐Leu plates. Possible autoactivation of the bait and the prey constructs was tested by cotransformation of the bait or prey vector with empty prey or bait vector, respectively.

GAL4 BD-fusion constructs for transactivation activity analysis, or GAL4 BD-fusion constructs and GAL4 AD-fusion constructs for yeast two-hybrid assay were transformed into the yeast strain, YD116, which harbors GAL1::URA3 and UAS_GAL4::lacZ as reporter genes. Yeast transformation was performed using the Frozen-EZ Yeast Transformation II^TM Kit (Zymo Research Corp., Irvine, CA, USA), according to the manufacturer’s instructions. Transformants were selected on synthetic minimal media lacking tryptophan (SM-Trp) or SM media lacking tryptophan and leucine (SM-Trp/-Leu).
For the yeast growth assay, transformants were streaked onto SM lacking tryptophan and uracil (SM-Trp/-Ura) or SM lacking tryptophan, leucine, and uracil (SM-Trp/-Leu/-Ura) and incubated at 30 °C for 3–5 days. A quantitative β-galactosidase assay using ONPG as a substrate was performed according to the methods described by Miller et al. [49 (link)]. The unit of β-galactosidase activity was then calculated using the formula 1000 × OD₄₂₀/(OD₆₀₀ × assay time in min × assay volume in mL). For the β-galactosidase filter assay, the transformants were analyzed using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as a substrate. The β-galactosidase filter assay was performed according to the Clontech Yeast Protocols Handbook (Clontech Laboratories, Inc., Mountain View, CA, USA). The reaction was carried out for 6 h.