2 Materials
1. Growth media: YPD media: yeast extract (1%), Bacto peptone
(2%), glucose (2%).
2. Selection media: 0.67% Bacto yeast nitrogen base without
amino acids, Glucose (2%) and all amino acids except tryptophan. 22 g/l of Bacto agar is added to obtain a solid selection
assay.
3. Plasmid pTSUK.
4. Saccharomyces cerevisiae strain SS111.
5. Electropulsator (see Note 1).
6. Digitized oscilloscope with a 5 MHz bandwidth.
7. PEF chamber is designed with two parallel flat stainless electrodes (1 cm width) with an interelectrode gap of 0.25 cm.
8. Laminar flow hood.
9. Incubator at 30
C.
10. Electrotransfer buffer (EB):10 mM Tris–HCl, pH 7.5,
270 mM sucrose, 1 mM MgCl 2 .
3 Methods (See Note 2)
3.1 Preparation
of Plasmids
1. Plasmid pTSUK is an E. coli–S. cerevisiae shuttle vector that
carries the CEN3 and ARS1 sequences and the TRP1 gene.
2. Plasmid DNA is extracted from DH5 alpha E. coli strain by
standard procedures and is purified before its use for GET by a
double centrifugation to equilibrium in cesium chloride–ethidium bromide. Ethidium bromide is cleaned out by using an
ion exchange resin. This procedure is observed to provide a
purified plasmid highly efficient for GET (see Note 3).
3.2 Electrotransfer
3.2.1 Preparation of Cells
1. Fresh cells are used even though frozen cells are also appropriate for gene electrotransfer (see Note 4).
2. Cells are grown at 30
C up to a density of about 10
7 cells per
ml. (see Note 5).
3. Cells are in the exponential growth phase. The growth medium
is YPD.
4. Cells are treated during 10 min with DTT 25 mM at room
temperature (see Note 6).
3.2.2 Gene
Electrotransfer
1. Cells are washed twice by centrifugation (1500 Â g, 5 min,
4
C) followed by resuspension in the low conductivity electropulsation buffer EB (see Note 7).
190
E. Meilhoc and J. Teissie
1. Growth media: YPD media: yeast extract (1%), Bacto peptone
(2%), glucose (2%).
2. Selection media: 0.67% Bacto yeast nitrogen base without
amino acids, Glucose (2%) and all amino acids except tryptophan. 22 g/l of Bacto agar is added to obtain a solid selection
assay.
3. Plasmid pTSUK.
4. Saccharomyces cerevisiae strain SS111.
5. Electropulsator (see Note 1).
6. Digitized oscilloscope with a 5 MHz bandwidth.
7. PEF chamber is designed with two parallel flat stainless electrodes (1 cm width) with an interelectrode gap of 0.25 cm.
8. Laminar flow hood.
9. Incubator at 30
C.
10. Electrotransfer buffer (EB):10 mM Tris–HCl, pH 7.5,
270 mM sucrose, 1 mM MgCl 2 .
3 Methods (See Note 2)
3.1 Preparation
of Plasmids
1. Plasmid pTSUK is an E. coli–S. cerevisiae shuttle vector that
carries the CEN3 and ARS1 sequences and the TRP1 gene.
2. Plasmid DNA is extracted from DH5 alpha E. coli strain by
standard procedures and is purified before its use for GET by a
double centrifugation to equilibrium in cesium chloride–ethidium bromide. Ethidium bromide is cleaned out by using an
ion exchange resin. This procedure is observed to provide a
purified plasmid highly efficient for GET (see Note 3).
3.2 Electrotransfer
3.2.1 Preparation of Cells
1. Fresh cells are used even though frozen cells are also appropriate for gene electrotransfer (see Note 4).
2. Cells are grown at 30
C up to a density of about 10
7 cells per
ml. (see Note 5).
3. Cells are in the exponential growth phase. The growth medium
is YPD.
4. Cells are treated during 10 min with DTT 25 mM at room
temperature (see Note 6).
3.2.2 Gene
Electrotransfer
1. Cells are washed twice by centrifugation (1500 Â g, 5 min,
4
C) followed by resuspension in the low conductivity electropulsation buffer EB (see Note 7).
190
E. Meilhoc and J. Teissie