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Cloning DNA into Plasmid Vectors and Sequencing.

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

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Cloning DNA into Plasmid Vectors and Sequencing.

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  1. Cloning DNA into Plasmid Vectors and Sequencing. ABE Workshop 2007 Josh Nelson 26 – Jun – 2007

  2. 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

  3. Steps in the Cloning Process Choose a vector • Isolate Protein

  4. Isolation of Protein • Amino acid sequencing • Purify protein • Sequence protein • Design primer • Run PCR • Result: DNA of interest PDI Isolate Protein • Start with PDI • Obtain sequence from database • Make primers • Run PCR

  5. Steps in the Cloning Process • Isolate Protein • Choose and Prepare Vector

  6. 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

  7. Steps in the Cloning Process • Isolate Protein • Choose and Prepare Vector • Transform intoHost

  8. 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

  9. Steps in the Cloning Process • Isolate Protein • Choose and Prepare Vector • Transform into Host • Extraction of Cloned Gene

  10. Extraction of Cloned Gene White Blue/white? • Choose colonies • 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

  11. Steps in Cloning the Process • Isolate Protein • Choose and Prepare Vector • Transform into Host • Extraction of Cloned Gene • Sequence

  12. Sequencing • Add primers to DNA • T7 upstream • T3 downstream • Dideoxynucleotides • Terminates synthesis • Fluorescent Dyes • Help in fast sequencing • Dyes different weights • Polyacrylamide Gel Electrophoresis

  13. Results of Sequencing • One was PDI 1 • White colony • One was vector • Base pairs between 1550 and 2300 • ccdB gene not expressed

  14. My Application • Shrimp aquaculture • Microbial community • Build up of ammonia • Clone gene which expresses desired trait

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