Standard techniques were used for DNA manipulation. Restriction enzymes were
purchased from New England Biolabs, except for PfoI, which was
purchased from Fermentas. Ligations were performed using T4 DNA ligase purchased from
Invitrogen. Both PCR-mediated site-directed mutagenesis and gene amplification for
cloning purposes were performed using either cloned Pfu Turbo DNA
polymerase (Stratagene) or KOD HotStart DNA polymerase (Toyobo, Novagen/EMD
Chemicals). Antarctic phosphatase (New England Biolabs) was used to treat symmetrical
ends of plasmids cut with a single restriction enzyme to prevent recircularization.
Plasmid propagation was carried out in Invitrogen MAX Efficiency DH5α bacteria
grown in lysogeny broth (LB) (Bertani 2004 (link))
supplemented with either 50–100 μg/ml ampicillin sodium salt or 10
μg/ml kanamycin sulfate purchased from Sigma-Aldrich. Bacterial transformants
were selected for on LB 2% agar plates supplemented with either 100 μg/ml
ampicillin sodium salt or 60 μg/ml kanamycin sulfate.
Plasmid construction details are provided insupporting information , File S1 . In general, we followed the strategy employed for mutagenesis
ofTRP1 , LEU2 , and URA3 during construction of the YXplac plasmids (Gietz and Sugino 1988 (link)). We used silent
mutations that preserve the amino acid sequence to mutagenize restriction sites found
in the open reading frame of the yeast-selectable auxotrophic marker genes
ADE2 , HIS3 , TRP1 , LEU2 , URA3 , ADE1 , and HIS2 (Table S1 ). As for the few sites occurring in the untranslated regions
of these genes, we used neutral changes that should not affect either transcription
initiation or termination. Oligonucleotides used for site-directed mutagenesis are
listed inTable S2 .
purchased from New England Biolabs, except for PfoI, which was
purchased from Fermentas. Ligations were performed using T4 DNA ligase purchased from
Invitrogen. Both PCR-mediated site-directed mutagenesis and gene amplification for
cloning purposes were performed using either cloned Pfu Turbo DNA
polymerase (Stratagene) or KOD HotStart DNA polymerase (Toyobo, Novagen/EMD
Chemicals). Antarctic phosphatase (New England Biolabs) was used to treat symmetrical
ends of plasmids cut with a single restriction enzyme to prevent recircularization.
Plasmid propagation was carried out in Invitrogen MAX Efficiency DH5α bacteria
grown in lysogeny broth (LB) (Bertani 2004 (link))
supplemented with either 50–100 μg/ml ampicillin sodium salt or 10
μg/ml kanamycin sulfate purchased from Sigma-Aldrich. Bacterial transformants
were selected for on LB 2% agar plates supplemented with either 100 μg/ml
ampicillin sodium salt or 60 μg/ml kanamycin sulfate.
Plasmid construction details are provided in
of
mutations that preserve the amino acid sequence to mutagenize restriction sites found
in the open reading frame of the yeast-selectable auxotrophic marker genes
of these genes, we used neutral changes that should not affect either transcription
initiation or termination. Oligonucleotides used for site-directed mutagenesis are
listed in
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