Yeast cell (EGY48 with p8op-lacZ reporter plasmid) transformation (for LexA library screen)
Method Article
Yeast transformation (for LexA library screen)
https://doi.org/10.1038/nprot.2006.418
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Yeast cell (EGY48 with p8op-lacZ reporter plasmid) transformation (for LexA library screen)
Solutions
10 ml 1X TE/LiAc:
1 ml 10X TE (pH 7.5)
1 ml 10X LiAc (pH 7.5)
8 ml ddH2O
= 10 ml
100 ml PEG/LiAc:
80 ml 50% PEG4000
10 ml 10X TE (pH7.5)
10 ml 10X LiAc
= 100 ml
1X TE buffer (pH 7.5):
3ml 10X TE (pH 7.5)
27ml ddH2O
= 30ml
(Adjust volumes of 1X TE and PEG/LiAC according to the number of transformations being done).
Library Scale: 150 ml in 500 ml flask
Incubate at 30 oC overnight (16-18 hr) with shaking (at 250 rpm) to stationary phase (OD600 greater than/equal to 1.5).
Library Scale: 1000 ml in 2L flask
Incubate at 30 oC for 3 hr with shaking (230 rpm). The OD600 will be 0.5 plus/minus 0.1.
Place cells in 50-ml tubes and centrifuge at 1500 rpm in floor centrifuge for 5 min at room temperature.
Discard the supernatant and resuspend cell pellets by vortexing in this volume of sterile TE or H2O: Small Scale: 25-50 ml
Library Scale: 500 ml
Pool cells in 50 ml tube and centrifuge at 1500 rpm in floor centrifuge for 5 min at room temperature.
Decant the supernatant.
Resuspend the cell pellet in this volume of freshly prepared, sterile 1xTE/LiAc: Small Scale: 1.5 ml
Library Scale: 8 ml
(NOTE: Cells must be used within about an hour after preparation.)
Library Scale: 100ml
Library scale (in 500 ml centrifuge tube)
Bait vector- 0.2 ug (small), 0.6 mg (library)
Library vector- 0.1 ug (small), 0.3 mg (library)
Herring testes carrier DNA- 0.1 mg (small), 20 mg (library)
(For simultaneous cotransfection, a molar ratio of 2:1 (Bait vector: Prey vector) is recommended). (Just prior to use, denature the carrier DNA by placing it in a boiling water for 20 min and immediately cooling it on ice)
Library Scale: 8 ml
Library Scale: 60 ml
Incubate at 30 oC for 30 min with shaking (200 rpm).
Add this volume of DMSO: Small Scale: 70 ul
Library Scale: 7 ml
Mix well by gentle inversion or swirling. Do not Vortex.
Heat shock for 15 min in a 42 oC water bath. Swirl occasionally to mix (large- and library-scale only)
Chill cells on ice for 1-2 min.
Centrifuge cells for:
Small Scale: 5 sec., room temperature - 14K rpm
Library Scale: 5 min., room temperature - 1500 rpm
Remove the supernatant.
Resuspend cells in this volume of 1 x TE: Small Scale: 0.5 ml
Library Scale: 10 ml
For large- and library-scale transformation:
--- Spread 100 ul of a 1:1,000, 1:100, and 1:10 dilution on SD-Ura-His-Trp plates (100 mm) for cotransformation efficiency controls.
--- Spread 1 ul (diluted in 100 ul of H2O) on SD-His-Ura and SD-Trp-Ura plates (100 mm) to check the transformation efficiency of each plasmid.
--- Spread the remaining transformation suspension on SD-Ura-His-Trp plates (200 ul per 150-mm Plates)
Incubate the plates at 30 oC for 3-5 days.
A. Cotransformation efficiency: count the colonies (cfu) growing on the SD-His-Trp-Ura dilution plate that has 30-300 cfu.
cfu/ug DNA = (Cfu x total suspension vol. (ul)) /
\(\(Vol. Plated \(ul)) x \(dilution factor) x \(amount.DNA used\(ug)\(Limiting plasmid)))B. The number of clones amplified:
cfu/ug x amt. Of library plasmid used (cfu/ug) = # of clones amplified.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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