NTU Undergraduates' research April 2014 - Biosciences | Seite 57

Abstract
To continue an ongoing investigation into the role of AGAP2 (ArfGAP(ADP
ribosylation factor GTPase activating protein), with GTPase domain, ankyrin repeat
and PH domain 2. Also known as PIKE-A), a mutagenesis experiment using an
AGAP2 pRK5 plasmid was planned. The aim was to design a set of oligonucleotides
which would recognise and anneal to the junction site in between the end of the pRK5
vector and the beginning of the AGAP2 sequence and to insert the a short sequence
coding for a protein tag (FLAG). A version of Stratagene’s QuikChange® SiteDirected Mutagenesis kit was used to perform the mutagenesis reaction. The PCR
product was then purified though a Dpn1 digest before being transformed into
competent DH5α cells (strain of Escherichia coli) which would then be minipreped to
isolate the mutated plasmid DNA. To check for the presence of the AGAP2 sequence
in the bacterial plasmid, a restriction enzyme digest was planned, using Not1 and Sal1
which would release the AGAP2 sequence from the plasmid. Upon attainment of
successful results, it was planned to then have the plasmid DNA sequenced to ensure
that the correct plasmid had been generated. Assuming the project was successful, the
FLAG tagged AGAP2 pRK5 plasmid could then be used to transfect into a
mammalian cell line for further investigations into the role of the AGAP2 protein.