Hi, I think you need to provide more details. The strain you are transforming carries the pRS426 plasmid but after transforming with a second plasmid, did you use double selection to maintain the pRS426 plasmid? It may very well be lost if you did not as the pRS426 plasmid is a 2 micron plasmid with no centromere sequence. As a result, the partitioning of the 2 micron plasmid between mother and daughter cell is unequal, unlike the partitioning of a chromosome, and if selection pressure isn't applied, the daughter cell may not receive any of the pRS426 plasmid at all.
On the other hand, a CEN plasmid like pRS416 would be more stable as it is attached to the spindle through its centromere sequence. However, I would still maintain -Ura selection as it is still less stable than a chromosome.
You might want to look up the original pRS426 paper (Christianson et al, 1992, Multifunctional yeast high-copy-number shuttle vectors, <I>Gene</I>) and the pRS300 series paper (Sikorski & Hieter, 1989, <I>Genetics</I>)
Originally Posted by Autophagator
I am performing my doctorate with yeast S.cerevisiae. Its a completely new organism for me, and now I am having troubles with him/her/she/he/it whatever.. Recently, I did transform a simple mutant (ura+) to create a double mutant. The simple mutant was ura+ ( with pRS426 plasmid) and grow normally in SC-ura plaques. However, after transformation (with other DNA and selection non relationated), the double mutant donít grow in liquid media SC-ura. Why?? I donít understand°° I did repeat the test several time, and still happens. Can anybody suggest me an idea, test, paper, link o something to know what happens here????
Thank a lot in advance.