Gene Therapy Lab Report

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Gene Therapy: Labs 1-3 Introduction Gene therapy is a current topic of much interest among researchers since often many genetic defects are the result of a single mutation on a gene. Ideally, if this could be fixed, then many genetic diseases could potentially be cured. This fact causes a considerable amount of experimentation to be conducted in this field. Based upon instructions and information found in our provided lab manuals we conducted this experiment. In this experiment, we attempted to do our own gene therapy experiment using the eukaryote Saccharomyces cerevisiae (baker’s yeast). This strain of yeast makes it an ideal candidate for doing gene therapy in both simplicity in form in higher eukaryotes, and a high efficiency in transformation;…show more content…
Using 500 microliters of solution 1 (sterile water) in an Eppendorf tube, we stirred each sample into solution. Each solution (2 Eppendorf tubes containing each strain) was centrifuged for approximately 4 seconds in a centrifuge. The goal of this is to pellet out the solute. Next, we resuspended the pellets in 500 microliters of solution 2 (100 mM LiOAc, 10mM Tris, 8.0, 1 mM EDTA) after decanting the supernatants. After resuspension, we spun the solution for 4 seconds and repeated the procedure of discarding the supernatant from the pellet. Once more we resuspended the pellet in solution 2, this time using only 100 microliters. After this, we split each resuspended pellet equally by putting 50 microliters in two new tubes. This made 4 total tubes that we labeled as: EAY235 TRP1, EAY235 LEU2, EAY431 TRP1, and EAY431 LEU2. 14 microliters of 5 mg/mL sspDNA (carrier DNA to boost transformation efficiency) was added to each of the four tubes. 6 microliters of pEA037 plasmid was added to the two TRP1 tubes and 5 microliters of pEAI27 DNA (linearized plasmid) was added to the two LEU2 labeled tubes. Next, 300 microliters of Solution 3 (100 mM LiOAc, 10 mM Tris, pH8.0, 1 mM EDTA, 40% PEG4000) were added to all 4 tubes and then vortexed until dissolved. These tubes were then incubated at 30 degrees Celsius for 30 minutes with agitation approximately every 10 minutes. Then the tubes were transferred to another water bath at 42 degrees Celsius for 15 minutes (this was to disrupt the membrane integrity to enhance the plasmid/DNA uptake). Once again, the solutions were centrifuged and discarded of supernatants (changing the tips between samples). The pellets were then resuspended in 50 microliters of solution 1 to aid in the removal of PEG 4000 in solution from the yeast). After this the solutions were vortexed. Solutions were centrifuged for 4 seconds
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