Creation of a PAX3 with a FLAG Tag Tool
Session Number
A01
Advisor(s)
Deborah Lang, University of Chicago
Location
B-110
Start Date
28-4-2016 11:05 AM
End Date
28-4-2016 11:25 AM
Abstract
The PAX gene makes proteins that bind to certain segments of DNA controlling the activity of that gene. The transcription factor PAX3 over-expresses in melanoma cell lines, affecting the development of melanocytes. The question remains then how phosphorylation of the PAX3 protein affects the stability of the PAX3 protein level. To answer this, the half-life of the PAX3 level must be examined. This project entails creating a tool for adding PAX3 with a FLAG tag which can be used in the future to examine the half-life of the PAX3 protein. PAX3 cDNA is amplified via PCR before being cut and then ligated with a cut portion of pcDNA-5’UT-FLAG vector to form a recombinant plasmid. Specifically, PCR and restriction enzyme digestion, ligation, transformation, and amplification form the recombinant plasmid. We check the accuracy by isolating the plasmid and performing diagnostics. If the project is successful and a tool has successfully been created, the isolated DNA may be sent to sequencing to do a final check on accuracy and the tool can be used for future projects. In the grand scheme of things, introducing a mutation to the epitopes that PAX3 is phosporylated to lets us see how phosphorylation affects turnover rate.
Creation of a PAX3 with a FLAG Tag Tool
B-110
The PAX gene makes proteins that bind to certain segments of DNA controlling the activity of that gene. The transcription factor PAX3 over-expresses in melanoma cell lines, affecting the development of melanocytes. The question remains then how phosphorylation of the PAX3 protein affects the stability of the PAX3 protein level. To answer this, the half-life of the PAX3 level must be examined. This project entails creating a tool for adding PAX3 with a FLAG tag which can be used in the future to examine the half-life of the PAX3 protein. PAX3 cDNA is amplified via PCR before being cut and then ligated with a cut portion of pcDNA-5’UT-FLAG vector to form a recombinant plasmid. Specifically, PCR and restriction enzyme digestion, ligation, transformation, and amplification form the recombinant plasmid. We check the accuracy by isolating the plasmid and performing diagnostics. If the project is successful and a tool has successfully been created, the isolated DNA may be sent to sequencing to do a final check on accuracy and the tool can be used for future projects. In the grand scheme of things, introducing a mutation to the epitopes that PAX3 is phosporylated to lets us see how phosphorylation affects turnover rate.