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
This TRAFO Protocol is for 2 Hybrid system screens
(We have done over 30 and still counting)
High Efficiency Transformation of a yeast strain requiring maintenance of a plasmid.
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is often more efficient to transform the library pool into a strain already containing the first plasmid (R.D. Gietz, unpublished data).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 ).
All solutions used in this protocol are described in the TRAFO Solutions Page
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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