Today we transformed the conjugative strain with the plasmid with kanamycin resistance containing the RFP reporter under the regulation of PY Promoter. We followed Protocol 3 (Electroporation).
After the transformation we plated the transformed cells in LB-AMP-KAN plates and let them grow at 37ºC for an O/N period.
Fabi and Léo
YeastGuard
Yeast experiments
<p style=”text-align:justify;”>To perform our tests, we digested the following minipreps: pJEN1+YFP, pJEN1+Lys, pDLD+YFP, pDLD+Lys with XbaI and PstI to connect to YEP vector. We also digested the plasmid YEP+Adh1-YFP to confirm the correct insertion of the part into YEP vector. The digestion showed that our new devices are ricght! =) But there is something wrong with the YEP+Adh1-YFP construction, it seams the digestion were incomplete.
We purified one digested fragment of each device: pJEN1+YFP (1781bp), pJEN1+Lys(1567bp), pDLD+YFP (1246bp), pDLD+Lys (1032bp). We ligated the digested fragments with YEP vector and transformed in competent E. coli.
We did miniprep of YEP+Adh1-lysozyme. Later we digested the plasmids to confirm the insertion of the Adh1-Lysozyme construction into YEP. We also digested 10 more plasmids YEP-Adh1-YFP again, this time we digested a little longer to try to avoid the incomplete digestion problem.
We prepares competent S.cerevisiaeand transformed competent yeasts with YEP+Adh1-YFP (Protocol 13) without confirming the correct ligation.
"
