Hello, I have trouble with dealing with yeasts. My problem is essentially 'yeasts are not growing'.
The yeast is S.cerevisiae, VL6-48 strain. I bought this strain from ATCC, and growing well in YPDA broth, so there is no problem until the transformation. I use LiAc/PEG method to transform the yeasts. And select the transformed colony using the auxotrophic method with Takara SD broth.
However, after transformation, they did not grow in SD broth or agar. The transformed colonies are small and growing painfully slow after subculturing. The transformed small colonies appear 2~3 days after transformation, but it takes 10 days or more to form the colony after subculturing (e.g streaking on the new SD agar plate.)
I checked they are not false-positive colonies, as I confirmed that the plasmids are present in the cells by PCR. Also, transformed colonies are growing well again in YPDA media.
What would be a reason for this phenomenon? Can it be a petit phenotype? Or, transformation efficiency is too low?
Please give me some advice.
Thank you.
Is it the same with transformants obtained with your plasmid without insert(s)?
@Herve Levesque, Thank you for your reply. I am trying with empty vectors.
Hi there,
Are you sure about your SD media? Did you check the starting strain actually grows normally on SC media?
Hi,
Please check your plasmid and SD media compatibility. Please describe, what is the composition of your media? As I know, many variants of SD medium (Takara) are available.
Dominique Liger , Dharmendra Nath Bhatt Thank you for your reply. My yeast strain is VL6-48. As I know, this strain cannot be grown in SD-His medium, so I choose it as the auxotrophic selection medium. Also regarding plasmid, it is pVC-604 YAC plasmid, it has HIS3 gene.
I did not check that whether the starting strain can grow on SD-his medium, I will check that. I think this would be necessary to confirm the authenticity of the strain.
Hi again, the purpose of my previous message was to verify that your minimal media is actually OK for your strain to grow when all the supplements are present (SC is synthetic complete medium which means it contains all aa bases and vitamins). In your case SC will be SD-His supplemented with his.