Yeast plasmid DNA miniprep problem

From: Zhang, Zhen
Date: 12 April 2012 16:32

Sorry for the non-ccp4 question. I am having trouble to miniprep plasmid from yeast. The plasmid is pCTCON2 and the yeast strain is EBY100. I am using Zymoprep Yeast Plasmid Miniprep II kit. I grow the culture in SDCAA medium to OD0.3 and spin down 1.5ml cells. The pellet was resuspended in solution 1 supplemented with Zymolyase and was incubated for 1 hr. I followed the manual exactly for the rest steps. However, I have never been able to recover any plasmid. There is nothing or smear in the agarose gel and there is no colony after transformation to DH5alpha after one overnight incubation at 37. The existence of the plasmid in the yeast has been confirmed by the ability to grow in the nutrition deficient medium SDCAA and the expression of plasmid protein in SGCAA medium was observed by FACS. I am wondering if someone here can shine some light on this issue. Any thoughts or suggestions are welcome.

 

Thank you.

 

Zhen

 

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From: Philipp Ellinger

Hi Zhen,

I also had problems with low amounts of plasmid from yeast using 5 ml o/n cultures. But when increasing the culture volume it worked:

Large-scale (0.5–1 l for obtaining preparative plasmid amounts) overnight  yeast cultures were harvested, washed in cold water once, and then resuspended in Zymolyase incubation buffer (0.1 M sodium phosphate buffer pH 7.4, 2 mM DTT, and 5–10 mg/ml of Zymolyase with 1.2 M sorbitol). The yeast cell wall was digested for 30 min at 37°C, cells were then lysed by alkaline lysis as (described in: Singh MV, Weil PA. A method for plasmid purification directly from yeast. Anal Biochem.2002;307:13–17). 0.5–1 µl of this preparation was used to transform chemically competent E. coli.

Cheers Phil