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Table 1. Centromeric and Episomal Plasmids drop-out powder (18) DNA [10 mg/mL salmon sperm DNA
lacking the appro- (Invitrogen) denatured at 100C for 10
Selection Replication
Name Marker Origin Control Digest
priate auxotrophic min and cooled on ice] were added.
marker. The appro- The cells were divided into appropriate
pRS41H hphNT1 ARS/CEN EcoRI: 1769; 3620 priate dominant drug aliquots and placed at -80C (no shock
pRS41K kanMX4 ARS/CEN HindIII: 1249; 3929 should be added after freezing). Usually, 10 L of competent
pRS41N natNT2 ARS/CEN XmaI: 1605; 3409 autoclaving of the cells were used for the transformation
pRS42H hphNT1 2 EcoRI: 1769; 4448
medium. of a centromeric or episomal plasmid
and 50 L of cells for the transfor-
pRS42K kanMX4 2 HindIII: 1249; 4757 mation of an integrative plasmid.
Yeast Transformation
pRS42N natNT2 2 XmaI: 1605; 4237 Thawed competent cells were added
Control digests to verify the plasmids can be made using the restric- The following to a sterile 1.5-mL tube containing the
tion enzymes indicated. The sizes of the resulting DNA fragments are protocol for yeast DNA (maximal 2 L plasmid DNA/10
given in base pairs. ARS, autonomously replicating sequence; CEN, transformation has L of cells). The suspension was mixed
centromeric sequence.
been previously well before a 6-fold volume of sterile
described (9). It is polyethylene glycol (PEG; 100 mM
The following media composition based on the lithium acetate method lithium acetate, 10 mM Tris-HCl, pH
was used for the double selection (20). Yeast cells were inoculated 8.0, 1 mM EDTA, 40% PEG 3350)
of an auxotrophic marker plasmid from a fresh pre-culture and grown was added. Cells were mixed again
together with a dominant drug resis- to a density of 0.50.7 A600 at 30C and incubated at room temperature
tance plasmid. The recipe was adapted (approximately 107 cells/mL) in YPD for approximately 30 min. Dimethyl
from the composition reported in the medium. Yeast cells were harvested sulfoxide (DMSO) was added to a
supplementary information (SGA by centrifugation (500 g for 3 min), final concentration of 10%. The cells
Analysis: Media) to Reference 19. The washed once with 0.10.5 volumes of were placed in a water bath at 42C
main change compared with standard sterile water, and once with 0.10.2 for 520 min (15 min works best with
synthetic complete (SC) medium was volumes of sterile SORB [100 mM most strains) and sedimented (23 min
the replacement of ammonium sulfate lithium acetate, 10 mM Tris-HCl, pH at 500 g). The cells were washed once
with monosodium glutamate as a 8.0, 1 mM EDTA/NaOH, pH 8.0, 1 M with YPD and resuspended in 3 mL of
nitrogen source. The medium contains sorbitol (special grade for molecular YPD. They were then incubated on a
20 g/L glucose, 1.7 g/L yeast nitrogen biology; Merck, Whitehouse Station, shaker for 46 h at 30C and spread on
base without ammonium sulfate and NJ, USA), adjusted with acetic acid to a plate with the appropriate dominant
amino acids (DifcoTM; BD, Franklin a pH 8.0]. The cells were resuspended drug selection marker.
Lakes, NJ, USA), 1 g/L monosodium in 360 L of sterile SORB per 50 mL Selection of drug resistance yeast
glutamic acid, and 2 g/L amino acid of cell culture and 40 L of carrier clones on plates often required replica
A B
Yeast origin of replication
(ARS/CEN or 2)
blue-white
selection
lacZ 5 end of integration site
(HIS3, LEU2, or URA3)
multicloning site
Figure 1. Features of the new episomal and centromeric plasmids (A) and the new integrative plasmids (B). The positions of the different selection
markers and multicloning and integration sites are indicated. Detailed maps are provided as supplementary material (Figures S1 and S2) that is available online
at www.BioTechniques.com. ARS, autonomously replicating sequence; CEN, centromeric sequence.