In this laboratory students perform three exercises as an introduction to the basics of handling and analyzing DNA. In the first, they expose circular plasmid, linear phage, and high molecular weight genomic DNA samples to a variety of physical, thermal, chemical, and enzymatic conditions that might be expected to affect DNA integrity. The DNA's are analyzed by electrophoresis on a group agarose gel. In the second, they pour and reconstruct a "Frankengel" (a gel containing sections with three different agarose concentrations) on which they run a DNA ladder in order to investigate the effect of gel pore size on DNA fragment mobility. In the third, they perform an UV spectrophotometric analysis of DNA from which they learn to make purity and concentration estimates.
Genomic and plasmid-encoded RAS2 genes from Baker's and brewing yeast strains will be analyzed using a shortened Southern blot procedure employing chemiluminescence. Unique parts of this procedure are the gravity-driven "upside down" transfer and a rotisserie-like hybridization oven, which greatly facilitate shortening the transfer time to 45 minutes and the hybridization step to 60 minutes. Chemiluminescence obviates problems associated with radioisotopes without sacrificing sensitivity: the plasmid-borne RAS gene can be visualized within 15 seconds and the single-copy yeast genomic RAS2 gene, within a minute. Topics for discussion related to this laboratory can include the concepts of RFLPs, evolutionary conservation of DNA sequences and the genetic basis of cancer.
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