Saccharomyces cerevisiae
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Information about each of the annotated genes:
Experiment to determine if the cloned PCR product is in the forward or reverse direction:
IDH1: First, it is necessary to digest the plasmids isolated from only one colony of bacteria. For IDH1, the restriction enzyme EcoR V is a good choice because it cuts at only one site within the gene insert as well as one site on the plasmid outside of the insert. Then this digestion is run on an agarose gel and probed with a radioactively labeled IDH1 gene. The size of the bands on the Southern blot indicates if the insert is in a forward or reverse direction. A band of approximately 355 bp means that the gene insert is in the forward direction while a band of approx. 758 BP means that the gene is in the reverse direction. If the insert is in the forward direction, then this colony of bacteria is appropriate to use for further experiment. If the insert is in the reverse direction, then another colony of bacteria must be chosen and tested to see if it has the gene insert in the appropriate direction.
The exact same process for IDH2, IDP1, IDP2, and IDP3 can be used for the same reasons, but different restriction enzymes must be used and bands of different sizes on the Southern blot will result.
IDH2: It is possible to use Bgl II. This will result in a band of approx. 793 BP if the insert is in the forward direction and a band of approx. 335 BP if the insert is in the reverse direction.
IDP1: It is possible to use BamH I. This will result in a band of approx. 807 BP for an insert in the forward direction and a band of approx. 534 BP for an insert in the reverse direction.
IDP2: It is possible to use Bgl II. This will result in a band of approx. 761 BP for an insert in the forward direction and a band of approx. 514 BP for an insert in the reverse direction.
IDP3: It is necessary to use two restriction enzymes because of the available restriction enzymes, none cut both within the gene insert and within the plasmid. Cla I will cut within the gene insert and BamH I will cut outside of the insert. A band of 522 BP indicates an insert in the forward direction and a band of 795 BP indicates an insert in the reverse direction.
For questions and comments elsellars@davidson.edu