Dear all,
I am trying to transform a URA3- mutant of K. marxianus strain with a plasmid containing Sacch URA gene. I checked whether the cells were URA3- or not by plating them on FOA. The URA3- did grow on FOA and wild type did not survive. I have tried multiple times using the Lithium acetate method on YNB plates without uracil added. But, have failed to get successful results. Only on one occassion I got colonies, but they lost the plasmid of interest and still managed to grown on YNB plates without URACIL. I followed the salmon sperm DNA protocol with PEG addition. Incubation at 30 degrees for 30 min followed by heat shock at 42 degrees. Can anyone suggest me a better protocol with higher transformation chances?
Have you tried electroporation? Sometimes that works better, especially for yeast.
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