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

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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

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Steps in the Cloning Process
Choose a vector

• Isolate Protein

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Isolation of Protein
PDI
Isolate Protein

• Start with PDI
• Obtain sequence from database
• Make primers
• Run PCR

• Amino acid sequencing
• Purify protein
• Sequence protein
• Design primer
• Run PCR
• Result DNA of interest

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Steps in the Cloning Process

• Isolate Protein
• Choose and Prepare Vector

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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

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Steps in the Cloning Process

• Isolate Protein
• Choose and Prepare Vector
• Transform into Host

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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

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Steps in the Cloning Process

• Isolate Protein
• Choose and Prepare Vector
• Transform into Host
• Extraction of Cloned Gene

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Extraction of Cloned Gene
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/white?
Blue
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Steps in Cloning the Process

• Isolate Protein
• Choose and Prepare Vector
• Transform into Host
• Extraction of Cloned Gene
• Sequence

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Sequencing

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

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Results of Sequencing

• One was PDI 1
• White colony
• One was vector
• Base pairs between 1550 and 2300
• ccdB gene not expressed

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My Application

• Shrimp aquaculture
• Microbial community
• Build up of ammonia
• Clone gene which expresses desired trait