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Cutting double-stranded DNA.
(a) The enzyme DNase cuts DNA at random sites.
Specific site cleavage.
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Video: Restriction http://www.dnalc.org/resources/animations/restriction.html
Specific restriction enzymes cut each DNA molecular at the same sequence site.
The ligation of two different pieces of DNA.
DNA ligase is an enzyme which remakes the phosphate diester bonds.
Video: restriction enzymes bio37 http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
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Agarose gel electrophoresis is used to separate DNA (and RNA) molecules according to size.
Ligation of a DNA fragment into a vector using the restriction endonuclease EcoRL and DNA ligase.
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The transformation of bacteria and the selection of cells with the recombinant vector.
Video: Key Steps of Molecular Cloning http://www.youtube.com/watch?v=sjwNtQYLKeU&feature=related
Video: Plasmid Cloning http://www.youtube.com/watch?v=acKWdNj936o http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Restriction map of pBR322 showing unique restriction sites, the ampicillin and tetracycline resistance genes, and the origin of replication.
Note how the cutting and insertion at a particular restriction site will result in loss of resistance.
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Selection for recombinant cells after transformation
Restriction map of pUC18/pUC19 showing the ampicillin resistance gene, the origin of replication, and the multiple cloning site within the lacZ gene.
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The selection of bacterial colonies containing a recombinant vector (pCU vector + DNA insert) by the selection of white colonies.
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The lytic life cycle of a bacteriophage
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The cloning of human DNA restriction fragments (
●
and
▲
) into a bacteriophage vector.
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The use of Agrobacterium tumefaciens to transfer DNA into plants.
Ti plasmid http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
An animation of a gene gun can be found at: http://www.hort.purdue.edu/hort/courses/HORT250/animations/Gene%20Gun%20Animation/Gen egun1.html
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