plasmid transformation using yeast

[davidfranco72]

Hi Everybody,

I had a question........
I`m working on a yeast display system using the yeast strain EBY100.
When I`m transforming the yeast with plasmid using competent yeast cells or electroporation device ......i have always colonies using the selective medium SDCAA.....even when the yeast strain is not transformed....
Did anybody encounter such a problem .....high background ?

The plasmid contains tryp- while the EBY100 strain does not.....so using the SDCAA media (that does not contain tryp) theoretically only the `transformed` yeast strain should grow and not the empty yeast strain........

please advice.
your help is highly appreciated.
Dave

[pigfarmer]

How do you know you don't have plasmid in the yeast?

[davidfranco72]

we received the EBY100 strain from two different sources ....what makes it unlikely that these yeast strain contain any plasmid

[pigfarmer]

Originally Posted by davidfranco72

we received the EBY100 strain from two different sources ....what makes it unlikely that these yeast strain contain any plasmid

So doesn't that make it more likely that they now have the plasmid in them, and something is wrong with whatever you are doing that makes you think you don't have plasmid in them? Yeast transformation with a amino acid marker is pretty damn good.

[davidfranco72]

to recapitulate..........

We received the EBY strains from two sources......
When we transform the yeast strains we can check the transformation efficiency by first doing some induction.......and then looking at the expression of a tag in our case cmyc tag by yeast......this works....

in the yeast strain that we electroporated but without adding some plasmids......they also grow in the selective media .....so it seems that the yeast strain contain tryp.....
After colony PCR to look at the presence of tryp.....
both show presence of tryp in their genome........
when inducing these yeast strain they don`t express the cmyc tag what indicates the absence of plasmid.......
The problem is that they still grow on selective media.....and shouldn`t .....
Could you give me suggestions ?
thanks
Dave

[jennifer.chang02]

Can you go back to your very original EBY strain that you received? It sounds like to me you might have gotten a contamination of your yeast strain with "in-the-wild" yeast. Check to see if the strain you electroporated can grow on -leu, -ura-, and -his. If it can grow on all of them, it's probably not the strain you think you have. "In the wild yeast" look like well, yeast, so it's really hard to know you have contamination until the strain doesn't behave the way it should.

[hairongxu88]

It seems that I have the same problem with you.

[hairongxu88]

It seems that I have the same problem with you. I want to know if the problem had solved? and how? I am seeking help urgently. Thanks advanced.

[hairongxu88]

Hello, davidfranco72,I saw your question about yeast EBY100 ,I want to know if the problem had
solved?and how? It seems that I have the same question with you.I don't know how to identify the
wild yeast which contain nothing plasmia. Thanks in advance

[hairongxu88]

anyone could help me?