All constructs used in this study were confirmed by DNA sequencing at the Oklahoma Medical Research Foundation DNA Sequencing Facility. The CnYPD1 gene was PCR amplified from cDNA (kindly provided by Dr Jan Fassler, University of Iowa) into a pTrcHis-TOPO vector using the pTrcHis TOPO® TA Expression Kit (Life Technologies, Carlsbad, California, USA) to construct the plasmid trcHis-CnYPD1. The pTrcHis-CnYPD1 plasmid served as a template for making a series of N-terminal truncation constructs (Δ5, Δ19, Δ43, Δ50, Δ60, Δ77, Δ83 and Δ100). The hypothetical phospho-accepting histidine residue of CnYpd1, H138, was mutated to a glutamine residue by site-directed mutagenesis PCR using the pTrc-CnYPD1 plasmid as a template. Three mutants were constructed using TrcHis-CnYPD1 as a template in order to determine which specific residues are involved in metal ion binding, E58A, E49A-E54A and D60A-E67A. CnYpd1-E58A was constructed using site-directed mutagenesis PCR. The mutants E49A-E54A and D60A-E67A were constructed using ligation-independent cloning (Scholz et al.2013 (link)).
Protocol detail
CnYPD1 Gene Constructs and Mutations
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization : University of Oklahoma
Top 5 similar protocols
Variable analysis
independent variables
- N-terminal truncation constructs (Δ5, Δ19, Δ43, Δ50, Δ60, Δ77, Δ83 and Δ100)
- Mutation of the hypothetical phospho-accepting histidine residue of CnYpd1, H138, to a glutamine residue
- Mutations E58A, E49A-E54A and D60A-E67A
- Not explicitly mentioned
- All constructs used in this study were confirmed by DNA sequencing at the Oklahoma Medical Research Foundation DNA Sequencing Facility
- The Cn
YPD1 gene was PCR amplified from cDNA (kindly provided by Dr Jan Fassler, University of Iowa) into a pTrcHis-TOPO vector - The pTrcHis-Cn
YPD1 plasmid served as a template for making the N-terminal truncation constructs - The pTrc-Cn
YPD1 plasmid was used as a template for the H138Q mutation - TrcHis-Cn
YPD1 was used as a template for the E58A, E49A-E54A and D60A-E67A mutations
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!