Modification of a Vector to Improve Screening Efficiency During Gene Cloning
Department
Biological Sciences
Major
Biological Sciences
Research Advisor
Aronstam, Robert
Advisor's Department
Biological Sciences
Funding Source
UMR cDNA Resource Center
Abstract
Ligation is frequently the most limiting and costly step during DNA cloning. One reason is the inability to detect successful double digestion of the vector. In the plasmid (pcDNA3.1+ 3xHA) only 21 nucleotides separate the EcoRV and Xhol restriction sites we use for gene insertion, making it impossible to identify plasmids that have been digested. To eliminate this problem, the plasmid was modified to contain a 1,407 nucleotide insert, between the two sites. Another problem has been ligation-induced destruction of the EcoRV site. To circumvent this, a primer was designed that re-creates the EcoRV site. This modified plasmid accelerates cloning by decreasing the likelihood of uncut plasmids reaching the ligation stage. Moreover, the EcoRI site permits "double sticky" EcoRI and Xhol/Xbal tagging reactions. This approach has wide applicability in the production of unique plasmids for specific cloning uses.
Biography
Jennifer Jacobi is a junior at the University of Missouri--Rolla and majoring in Biology. She is the daughter of Susan Sebacher and Robert Jacobi and is from St. Charles, MO. On campus she is the president of Helix, a member of Phi Sigma, and Outreach Chair at the Newman Center. She is employed at the UMR cDNA Resource Center and is also working on a research project with two of her peers. After graduation Jennifer plans to attend graduate school to earn a PhD in Biology.
Research Category
Natural Sciences
Presentation Type
Poster Presentation
Document Type
Poster
Presentation Date
12 Apr 2006, 1:00 pm
Modification of a Vector to Improve Screening Efficiency During Gene Cloning
Ligation is frequently the most limiting and costly step during DNA cloning. One reason is the inability to detect successful double digestion of the vector. In the plasmid (pcDNA3.1+ 3xHA) only 21 nucleotides separate the EcoRV and Xhol restriction sites we use for gene insertion, making it impossible to identify plasmids that have been digested. To eliminate this problem, the plasmid was modified to contain a 1,407 nucleotide insert, between the two sites. Another problem has been ligation-induced destruction of the EcoRV site. To circumvent this, a primer was designed that re-creates the EcoRV site. This modified plasmid accelerates cloning by decreasing the likelihood of uncut plasmids reaching the ligation stage. Moreover, the EcoRI site permits "double sticky" EcoRI and Xhol/Xbal tagging reactions. This approach has wide applicability in the production of unique plasmids for specific cloning uses.
