The YEP digestions that we did yesterday seem incomplete again. Because of that we didn’t digested the new devices but we did colony PCR to confirm the correct insertion of them (pJEN1+YFP, pJEN1+Lys, pDLD+YFP and pDLD+Lys) in YEP. We also inoculated the same colonies in liquid LB+AMP media as a backup.
GEL
Only the part pDLD+Lys was confirmed to be correctly placed in the YEP vector. Considering we don´t have enough time to repeat the experiments till the wiki freeze, we had decided to proceed only with the pDLD+Lys construction and give up on the other constructions with the promoters.
So we did miniprep of the right pDLD+Lys colony and transformed in competent yeast.
The transformed YEP+Adh1-YFP yeast grew!!! =) We tried to see its fluorescence under blue light but it didn´t work. =(
Because of this result we decided to test the plasmids used to transform the yeasts and check if they really have our construction. We did PCR of the 10 YEP+Adh1-Lysozyme used plasmids and chose 10 more bacterial colonies to screen. We confirmed one correct plasmid (nº7)!!
GEL
We also digested 8 YEP+Adh1-YFP plasmids but the digestion with XbaI and PstI presented the same strange pattern so we decided to digest with another restriction enzyme: EcoRI. Unfortunately the digested fragments’ size seams to be wrong. =(
GEL
GEL
According to the PCR results, we also transformed competent yeasts with Adh1+Lys.
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