Plasmid Shuffle Method

The purpose of plasmid shuffle is to start with one plasmid in a cell, have two plasmids in the same cell, and eventually wind up with only the second plasmid in the cell.(see original paper)

1) At the beginning, there is a mutant strain of cells that is his- leu- and must be grown in media that contains both histadine and leucine. There are two plasmids that will be introduced into the strain as well.

His- Leu- strain:

His- Leu- strain grown in complete media:

2) Next, transform the bait plasmid (called pGAL4-bind) that provides the GAL4 binding domain and a his+ allele.

3) Now transform in the prey plasmid (pGAD) and select for His+ Leu+ cells.

4) In the yeast two-hybrid system, screen for cells that turn blue.

5) Now grow the cells in complete media for a few hours and then plate the cells on complete media.

6) Screen for cells that can grow in leu- media but cannot grow in his- media. These cells must contain only the pGAD plasmid:

7) Isolate the pGAD plasmid and transform into bacteria using ampicillin as the antibiotic.

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