2 Hybrid System TRAFO Protocol

 

 

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Note: Please cite

Agatep, R., R.D. Kirkpatrick, D.L. Parchaliuk, R.A. Woods, and R.D. Gietz (1998) Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online ( http://tto.trends.com ).

For complete instructions on how to do a two hybrid screen see the following references.

1. Gietz, R.D., B. Triggs-Raine, A. Robbins, K.C. Graham, and R.A. Woods (1997) Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system. Mol Cell Biochem 172:67-79.

2. Parchaliuk, D.L., R.D. Kirkpatrick, R. Agatep, S.L. Simon and R.D. Gietz (1999) Yeast two-hybrid system screening. Technical Tips Online ( http://tto.trends.com ) (accepted).not on line yet

SEE TRAFO REFs

 

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This TRAFO Protocol is for 2 Hybrid system screens
(We have done over 30 and still counting)

 

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High Efficiency Transformation of a yeast strain requiring maintenance of a plasmid.

 

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The two-hybrid system and other similar genetic screens in yeast involve the use of two different plasmids in a single yeast cell. One plasmid often contains a cloned gene or DNA sequence while the other plasmid contains a library of genomic or cDNA. While both plasmids can be co-transformed into a single yeast cell, it is often more efficient to transform the library pool into a strain already containing the first plasmid (R.D. Gietz, unpublished data).

Table 1 & Discussion ).

 

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All solutions used in this protocol are described in the TRAFO Solutions Page

 

 

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1. Inoculate the yeast strain containing the first plasmid into the appropriate volume of the appropriate SC-omission medium in a flask and incubate at 30 ^o C overnight.

   For each different Scale up use the appropriate size of culture
    

   TRAFO SCALE	10 X	30 X	60 X
   Culture Size	25 mls	50mls	100 mls