Student Scholars Day

Subcloning of a pkN cDNA from D. melanogaster

Delorean flies contain a wing mutation which is caused by a transposon in the protein kinase N gene. The overall purpose of this experiment was to insert the pkN cDNA RH37580 into the expression vector pCaSper, which would then be inserted into D. melanogaster embryos. Adult flies with the expression vector would be heated to activate the heat-shock sequence of the pCasper vector and RH37580 sequence to correct the wing mutation. Using PCR and specific primers, the open reading frame of RH37580 was amplified to provide enough genetic material for ligation into the pCaSper vector. The cDNA material was then ligated into vector pGem-T Easy. The vector was successfully transformed into bacterial cells, and blue-white screening was used to identify which colonies contained the pGem-T Easy vector with the RH37580 cDNA. PCR from direct bacterial samples was used to identify the positive clones, again for amplification. Ligation was then attempted to insert the cDNA into the pCasper vector.