Plasmid carrying the c-myc3–tagged versions of wild-type and mutated alleles of the ScELP3 gene [ScELP3-(myc)3] are based on the centromeric plasmid p424TDH (60). The gene was amplified by PCR from genomic DNA using yeast strain FFY3t (ELP3-(myc)3::SpHIS5) as a template. The PCR fragment was cloned into p424TDH using Eco RI and Xho I to give pON34 (ELP3-(myc)3). pON34 served as a template to generate mutated elp3-(myc)3 alleles by using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) to construct the mutated versions elp3-H110A-(myc)3, elp3-C118A/C121A-(myc)3, elp3-V119A/Y120A-(myc)3, (elp3-Y136A-myc)3, elp3-R232A-(myc)3, elp3-R269A-(myc)3, elp3-H271A-(myc)3, and elp3-W341A-(myc)3. The other mutations were transferred from plasmids of the GAL-driven overexpression system. Plasmids carrying elp1_Δ811-853 or elp3_Δ1-93 were generated using the In-Fusion HD Cloning Kit (Takara Bio USA Inc.). For elp1_Δ811-853, two fragments were amplified by PCR from genomic DNA using yeast strain UMY2893. Fragment 1 was amplified using ELP1_pUC19-F (5′-CGACGGCCAGTGAATTCATGGTTGAACATGACAAGAGTG-3′) and ELP1_d811-R (5′-TATTTTGTTGACTTTATCTTCCGACAAACAGGAAA-3′). Fragment 2 was amplified with the following oligonucleotides: ELP1_d853-F (5′-AAAGTCAACAAAATATGCGATGCTGTGC-3′) and ELP1_pUC19-R (5′-ATTACGCCAAGCTTGCATGCCTGCTGCTCAAAAATCAACAATATGACTCTTAGGG-3′). For elp3_Δ1-93, the first fragment was amplified using Elp3-pUC19-F (5′-CGACGGCCAGTGAATTCATCACTTATAACGTTGTGTT-3′) and Elp3_d2-93_R (5′-CACTGCAATACCCGACATCTTTGTCAGGGTGTTCTTCGA-3′) from pRZ130 (pJet200 XbaI/XhoI + PELP3-elp3_E485A-3myc- SpHIS3-TermELP3) and the second fragment was amplified using Elp3_d2-93_F (5′-TCGGGTATTGCAGTGGTAGC-3′) and Elp3-pUC19-R (5′-CCAAGCTTGCATGCCTGCTGAATCTTCAAAATATATGTACGT-3′) from pRZ135 (pJet200 XbaI/XhoI + PELP3-elp3_K86A-K88A-3myc-SpHIS3-TermELP3). Both fragments were cloned in the linearized (with EcoRI and SphI, respectively) plasmid pUC19 (61).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.