Hello,<br><br>I am doing yeast (<i>S. cerevisiae</i>) transformation for the first time. <br><br>My current work flow is:<br><br><div style="margin-left: 40px;">ligation of yeast plasmid (pYC2/NT A) and gene of interest --> "1st"
<i>E. coli </i>transformation --> plasmid extraction (check with enzyme digestion) --> yeast (INV<i>Sc</i>1) transformation --> plasmid extraction --> "2nd" <i>E. coli </i>transformation --> plasmid extraction (check with enzyme digestion) --> yeast (INV
<i>Sc</i>1) transformation --> over-expression of the protein<br></div><br>I have tried few method including Gietz, R.D. & Schiestl, R.H. <u>Quick and Easy Yeast Transformation Using the LiAc/SS Carrier DNA/PEG Method
</u>. <i>Natural Protocols</i> (2007), and find the transformation yields are very satisfying.<br><br>But I am facing problem in plasmid extraction from yeast where I am getting low <i>E. coli </i>transformants. The yields are low in term of lower in number of colonies and smaller colonies, compare to 1st
<i>E. coli </i>transformation. <br><br>For rapid isolation of plasmid from yeast, I am currently using both original and lab protocol which modified from Ausubel F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., & Struhl, K.
<u>Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology (5th ed)</u>, <i>New York: John Wiley & Sons, Inc</i>. (2002), and Robzyk, K., and Kassir, Yona., <u>A simple and highly efficient procedure for rescuing autonomous plasmids from yeast
</u>, <i>Nucleic Acids Research</i> (1992).<br><br>I would like to ask:<br><ol><li>is it normal for the "2nd" transformation having lower yield compare to the "1st"?</li><li>if not, any other suggestion of plasmid extraction? i am using beads in the extraction.
</li><li>is the "2nd" <i>E. coli </i>transformation necessary? this step was include because most of the protocol i read do so, but i do not understand why. Can somebody brief explain? or suggest a reading material?
<br></li></ol>Thank you very much.<br><br>Lau<br>
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