Transcript 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?
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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
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Amino acid sequencing
 Start with PDI
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Purify protein
 Obtain sequence from
database
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Sequence protein
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Design primer
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Run PCR
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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
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pCR 4 – TOPO
• Small plasmid
• LacZ – ccdB
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Selective for ccdB gene
Insert PCR Product
• A overhang
• Vector has T overhang
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Ligation
• Topoisomerase
Steps in the Cloning Process
1.
2.
3.
Isolate Protein
Choose and
Prepare Vector
Transform into
Host
Transforming into Expression Host
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E. Coli Cells
• Soak in CaCl2
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Make them
competent
Insert vector
• Heat shock
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Spread on culture
plate
• Kanamycin added
for selection
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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
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Choose colonies
Blue/white?
White
• White
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Place in LB medium
• Contains Ampicillin &
Kanamycin
• Incubate
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Isolate cloned gene
• Cell pellet
• Gene pellet
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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
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Add primers to DNA
• T7
• T3
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upstream
downstream
Dideoxynucleotides
• Terminates synthesis
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Fluorescent Dyes
• Help in fast sequencing
• Dyes different weights
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Polyacrylamide Gel
Electrophoresis
Results of Sequencing
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One was PDI 1
• White colony
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One was vector
• Base pairs between
1550 and 2300
• ccdB gene not
expressed
My Application
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Shrimp aquaculture
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Microbial community
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Build up of ammonia
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Clone gene which expresses desired
trait