IDH Gene Information
"This web page was produced as an assignment for an undergraduate course at Davidson College."
The first step of the Davidson College Molecular Biology Laboratory Project for this spring semester is to clone the five yeast IDH genes using PCR and then express the proteins in bacteria. The molecular weight of the IDH proteins will then be determined using a western blot and the yeast gene will be used as a probe for other yeast orthologs. Lastly, enzyme assays will be performed on the proteins to determine if they are functional in a prokaryote (Campbell, 2002).
The buttons below contain the following information about the 5 IDH genes:
Links to gene and amino acid sequences, accession numbers, six-cutter restriction sites, coenzymes, and fusion protein sequences. For more information about designing primers, designing DNA digestion with restriction enzymes in order to check the orientation of the cloned IDH genes, and the progress of the experiment, see below.
First, individuals in the class purified yeast genomic DNA using a Qiagen DNeasy Kit. Next we had to design and order the primers for PCR.
How do you design primers?
Follow this link for some helpful hints or click above for the explanation of exactly how it was done in this lab.
The following week the isolated DNA from the 5 IDH genes was amplified using PCR. Since the Mg+ concentration is a variable in PCR, we had to make a few modifications to the concentration, but after a few tries, good DNA yield was verified by gel electrophoresis. Take a look at our gels and learn more about the importanct of Mg+ in PCR.
The next step was to clean the PCR products, ligate the PCR products into an expression vector (pQE-30 UA) (Qiagen, 2002), and then transform the ligation into the JM109 strain of E. Coli (Promega, 2000). Click here for the transformation protocol that was used.
This week we will perform a miniprep of the plasmid DNA, digest the plasmids with the appropriate restriction enzymes, and verify the orientation of the PCR product with gel electrophoresis. Click here to see the design of this verification experiment.
Picture of yeast from TIGR Saccharomyces cerevisiae Gene Index (ScGI) webpage. <http://www.tigr.org/tdb/scgi/>
Saccharomyces Cerevisiae is a model organism for genetic and molecular research due to its quick replication cycles, abundance, and low cost among many other reasons. It was also one of the first organisms to have its entire genome sequenced.
Complete Genome of Saccharomyces Cerevisiae