Cloning dna into plasmid vectors and sequencing
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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

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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

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

Steps in the Cloning Process

Choose a vector

  • Isolate Protein


Isolation of protein

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


Steps in the cloning process1

Steps in the Cloning Process

  • Isolate Protein

  • Choose and Prepare Vector


Choosing and preparing the 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 process2

Steps in the Cloning Process

  • Isolate Protein

  • Choose and Prepare Vector

  • Transform intoHost


Transforming into expression 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 process3

Steps in the Cloning Process

  • Isolate Protein

  • Choose and Prepare Vector

  • Transform into Host

  • Extraction of Cloned Gene


Extraction of cloned gene

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


Steps in cloning the process

Steps in Cloning the Process

  • Isolate Protein

  • Choose and Prepare Vector

  • Transform into Host

  • Extraction of Cloned Gene

  • Sequence


Sequencing

Sequencing

  • Add primers to DNA

    • T7 upstream

    • T3 downstream

  • Dideoxynucleotides

    • Terminates synthesis

  • Fluorescent Dyes

    • Help in fast sequencing

    • Dyes different weights

  • Polyacrylamide Gel Electrophoresis


Results of sequencing

Results of Sequencing

  • One was PDI 1

    • White colony

  • One was vector

    • Base pairs between 1550 and 2300

    • ccdB gene not expressed


My application

My Application

  • Shrimp aquaculture

  • Microbial community

  • Build up of ammonia

  • Clone gene which expresses desired trait


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