I have problems in yeast transformation. Our lab uses the Y2H system from Clonetech. I followed the transformation protocol from the kit to transform my bait plasmid with pGADT7 vector plasmid to test the bait. However, no colonies formed on Leu-Trp plates after 4 days. Both positive and negative control grow normal colonies. The yeast cells and pGADT7 vector plasmid were from -80 and -20, respectively. My other labmates use the same protocol and they successfully completed their experiments.
Why did this initial transformation not work for me? Any idea
I am using -2 minimal yeast medium. Media is ok nothing wrong with it because it giving me colonies for other protein interactions for other genes.
But i am worried about my 2 genes why they are not giving me colonies both positive, negative control and other genes giving me colonies why not these two genes.
Positive control : pGADT7 (empty vector) + BD-53
Negative Control: pGADT7 (empty vector) + BD lambda
pGADT7 = Prey Plasmid (constructed with my genes of interest) + BD
I am using Yeast Gold strain Competent cells 100 ul (Stored at -80 freshly prepared).
I am wondering either these proteins may have some toxic properties or not.
1. One gene contain WSC domain proteins
2. Other genes contain KNR4/SMI1 proteins
If you can provide me some information about these proteins it will be good for me then I have the plan to make mutants and then screen out. Until now what I understand KNR4 proteins have some toxic effects but I am not sure about it. I need to study more about that.
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