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Upon insertion of a known gene sequence into a known plasmid vector, two potential outcomes are possible. Ideally, the gene inserts in the correct direction so that the start codon is at the "left" side of the vector, i.e., diagramatically oriented in conjunction with the PCR insert site in this case, the pQ-30UA vector. The other alternative is that our sequence inserts in the opposite direction, producing a "backwards" gene that will not express correctly because it cannot be read with the start codon at the end of the gene, i.e., the polymerase will not translate 3'-5'.
We must then design an expermined in order to determine if each inserted gene has inserted into the plasmind in the correct orientation. We must determinewhere known restriction sites are located one the gene and in the plasmid and splice the plasmid accordingly.. By cutting a known plasmid with a restriction enzyme that will splice both the sequence and a splice site within the plasmid, we can use gel electrophoresis to figure out length size. If the insert has inserted correctly, we will expect "size a" and a big band of lthe rest of the plasmid. If it inserted incorrectly, we will expect "size b" and a big band of the rest of the plasmid.
The chart below describes the restriction enyzmes and expected band sizes for each of the five genes we are using in lab that are described in the description of five yeast genes. The band sizes are based on information from the Sacchromyces Genome Databases summed to the appropriate location of restriction site on our plasmid, and are linked appropriately.
|S. cerevisiae gene||IDH1||IDH2||IDP1||IDP2||IDP3|
|Restriction enzyme||EcoRV||EcoRV||BamHI||BglII||ClaI (only in gene) & EcoRV on plasmid|
|If inserted correctly, size of expected band using pQ-30 UA vector||356||806||800||510||510|
|If inserted incorrectly (backwards), size of expected band using pQ-30 UA vector||759||336||527||760||785|
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