Type of Submission
Poster
Keywords
Plasmid Vectors, Protocadherin-19, Ncadherin, Neurodevelopmental Proteins
Proposal
Throughout this semester, work has been done to generate copies of the human genes for protocadherin-19 (PCDH-19), neural cadherin (NCAD), receptor-like tyrosine kinase (RYK), and a recombinant protein of Green Fluorescent Protein (GFP) with a Protocadherin-19 signal peptide that would create a form of GFP secreted from the cell. These cloned vectors would then be able to be used to study whether disruptions of interactions between these proteins leads to a female-specific form of epilepsy. In order to achieve this, the Platinum Taq DNA Polymerase High Fidelity system was used to generate many copies of the template target DNA. These copies were then identified using gel electrophoresis and isolated using the NucleoSpin Gel and PCR cleanup kit. A restriction digest was done to create ends compatible for insertion into a target vector digested with the same enzymes. Results of the digest were verified using gel electrophoresis. Inserts were then ligated to the plasmids using the T4 DNA ligase system and transformed into DH5𝞪 Competent cells. The plasmid contained a kanamycin resistance gene which was used to identify successfully transformed cells. The plasmids were then isolated using the NucleoSpin Plasmid Kit. Data from gel electrophoresis at intermediate steps provides preliminary evidence of successful cloning of each construct for protein expression. This will allow protein interactions to be tested in a mammalian cell culture system.
Creative Commons License
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Development of Plasmid Vectors of Neurodevelopmental Proteins
Throughout this semester, work has been done to generate copies of the human genes for protocadherin-19 (PCDH-19), neural cadherin (NCAD), receptor-like tyrosine kinase (RYK), and a recombinant protein of Green Fluorescent Protein (GFP) with a Protocadherin-19 signal peptide that would create a form of GFP secreted from the cell. These cloned vectors would then be able to be used to study whether disruptions of interactions between these proteins leads to a female-specific form of epilepsy. In order to achieve this, the Platinum Taq DNA Polymerase High Fidelity system was used to generate many copies of the template target DNA. These copies were then identified using gel electrophoresis and isolated using the NucleoSpin Gel and PCR cleanup kit. A restriction digest was done to create ends compatible for insertion into a target vector digested with the same enzymes. Results of the digest were verified using gel electrophoresis. Inserts were then ligated to the plasmids using the T4 DNA ligase system and transformed into DH5𝞪 Competent cells. The plasmid contained a kanamycin resistance gene which was used to identify successfully transformed cells. The plasmids were then isolated using the NucleoSpin Plasmid Kit. Data from gel electrophoresis at intermediate steps provides preliminary evidence of successful cloning of each construct for protein expression. This will allow protein interactions to be tested in a mammalian cell culture system.
