I have Saccharomyces cerevisiae strains that are deficient in some plasma membrane proteins that makes them sensitive to any stress and in the same way to Lithium acetate too. While using the LiAc/PEG method (Gietz & Schiestl, 2007 Nat protocol) i did not get any transforments. I repeated the experiment with different concentrations of the LiAc to optimise it. I used the a range of LiAc from 2-100mM of LiAc. Still i did not get any transforments. I have used this protocol before with other strains and it worked very well.
I have used the fresh buffers every-time. I have used freshly made PEG. I have also used freshly aliquoted ssDNA. I am trying to disrupt a region in genome and integrate a gene of my interest. To PCR amplify this gene I am using the primers having 40bp overhang complementary to targeted region. If anyone can help me with transformation of these sensitive strains then it will be great help for me. Thanks
Hi there,
Did you run exactly the same experiment with a more robust strain as a control? If not you can't be 100% sure the problem comes from the protocol (it might come from the experiment itself...).
I would also suggest to try other transformation methods like electroporation or spheroplast.
Hi Dominique Liger thank you so much for replying.
I have not used any robust strain as a control. I will repeat the experiment with a control this time.
Can you please give me a link to electroporation and spheroplast methods, that have worked well for you? Thank you for your suggestions.
I always use the Zymo Frozen EZ Yeast Transformation II, but I think it has lithium chloride in it. The only protocol I know of that has no lithium is speheroplasting:
https://openwetware.org/wiki/Spheroplast_Transformation
Pamela A Marshall thank you so much for your response. I will try spheroplast transformation with my strains.
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