Hi, I was lately trying to do a rescue of a gene knock out I did in yeast. I got a good phenotype from the knockout but could not restore the knockout by cloning the gene back in. Now I am trying to figure out why.

 

Originally I cloned my gene into a yeast vector which uses a uracil marker, but was instructed to remove the uracil marker from my knock out first since it already had that one. So I cultured the yeast in medium with 5'-floroorotic acid which selects against ura3 producing cells. This was supposed to remove the marker, so I cultured the colonies in rich media and then did a transformation using the LiAc method. At that point I believed I had my knock out with the gene cloned into the vector. Is that reliable? Currently I am unable to purify the plasmid to check it by purification and digestion.

Let's start with the beginning. What precisely do you start with? A knockout yeast and a vector with the complete gene that was knocked out? And there is URA3 in the vector as well as the yeast chromosome?

How was the knock out constructed. Was it a plasmid insertion (i.e. plasmid with URA3)?