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Cloning DNA into Plasmid Vectors and Sequencing. ABE Workshop 2007 Josh Nelson 26 – Jun – 2007 What is Cloning? Making multiple identical copies of a gene This can be placed into a vector This in turn can be placed into a host bacteria Modern plasmid vectors are designed to make the process fast and easy Inclusions of antibiotics and other screening methods ensure correct gene cloning Steps in the Cloning Process Choose a vector 1. Isolate Protein Isolation of Protein Isolate Protein PDI Amino acid sequencing Start with PDI Purify protein Obtain sequence from database Sequence protein Design primer Run PCR Result: DNA of interest Make primers Run PCR Steps in the Cloning Process 1. 2. Isolate Protein Choose and Prepare Vector Choosing and Preparing the Vector pCR 4 – TOPO • Small plasmid • LacZ – ccdB Selective for ccdB gene Insert PCR Product • A overhang • Vector has T overhang Ligation • Topoisomerase Steps in the Cloning Process 1. 2. 3. Isolate Protein Choose and Prepare Vector Transform into Host Transforming into Expression Host E. Coli Cells • Soak in CaCl2 Make them competent Insert vector • Heat shock Spread on culture plate • Kanamycin added for selection Incubate Steps in the Cloning Process 1. 2. 3. 4. Isolate Protein Choose and Prepare Vector Transform into Host Extraction of Cloned Gene Extraction of Cloned Gene Choose colonies Blue/white? White • White Place in LB medium • Contains Ampicillin & Kanamycin • Incubate Isolate cloned gene • Cell pellet • Gene pellet Run gel to confirm you cloned correct gene Blue Steps in Cloning the Process 1. 2. 3. 4. 5. Isolate Protein Choose and Prepare Vector Transform into Host Extraction of Cloned Gene Sequence Sequencing Add primers to DNA • T7 • T3 upstream downstream Dideoxynucleotides • Terminates synthesis Fluorescent Dyes • Help in fast sequencing • Dyes different weights Polyacrylamide Gel Electrophoresis Results of Sequencing One was PDI 1 • White colony One was vector • Base pairs between 1550 and 2300 • ccdB gene not expressed My Application Shrimp aquaculture Microbial community Build up of ammonia Clone gene which expresses desired trait