I have to transform two plasmids into yeast. One is 8kb with trp marker and other is 14 kb with ura marker. both are 2micron. I used Gietz et al method.
First, i tired tansforming both plasmids at same time and after plating on trp/ura plates, no colonies were observed.
Secondly, I prepared competent cell containing 8kb plasmid (trp marker) and tired transforming 14 kb plasmid (used 500ng). Still no colonies were observed. But the controls were perfectly alright. i could see lawn of cells in Trp drop out plate (POSITIVE CONTROL). As negative control, cells were plated in ura drop out plate and no colonies were observed. How could I solve this issue?
Maybe try putting in the ura marker plasmid first? It's a larger vector so it will be less efficient so if you can get that one to work then you can add in your trp vector.
For your putting the ura vector into your trp cells, did you try the control of just the ura vector? That will let you know if perhaps that part needs optimizing.
Double transformations can be inefficient.
I have transformed successfully before ura plasmid of 15kb. It worked perfectly fine with single selection marker.
I agree double transformation could be less efficient. That's the reason for the second time, i tried preparing competent cell of yeast containing 8kb plasmid in selective media.
I could do it other way round, but it will be laborious as i have many mutant version of 14kb plasmids which i need to transform.
thanks for your comment Amy!!
Hi there,
Getting no clone from transformation with 500ng of plasmid is not normal. Are you sure of the plates you are using (ie. did you grow a ura+ strain on it as a control?)? Are you sure of the plasmid integrity ? Are you sure of the reagents you use? If in doubt prepare them fresh...
i have no doubt in reagents, i use them routinely for my experiments. I would try transforming only ura plasmid (14 kb) into yeast and see the outcome. thanks Dominique Liger
I am coming back with some updates on the questions i asked before.
1. I tried transforming 14kb plasmid with ura marker into yeast; it worked perfectly well and i had several clones on the selection plate.
2 . For second plasmid tarnsformation, I prepared new competent cells containing 8kb plasmid with trp marker and transformed 14kb plasmid with ura marker. I increased the incubation time to 2hrs. i had colonies on plate but the efficiency is too low as compared to single plasmid transformation. Nevertheless, few colonies served my purpose.
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