Application in Molecular Cloning David Shiuan Department of

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Application in Molecular Cloning David Shiuan Department of Life Science, Institute of Biotechnology and

Application in Molecular Cloning David Shiuan Department of Life Science, Institute of Biotechnology and Interdisciplinary Program of Bioinformatics National Dong Hwa University

Molecular Cloning In order to have enough DNA to work with for a single

Molecular Cloning In order to have enough DNA to work with for a single gene or sequence, you must have a way to “clone”, or reproduce many exact copies of that gene. This is called “molecular cloning” Gene of interest

Plasmid Cloning Vector 1. Small 2. Stable in the chosen host – usually E.

Plasmid Cloning Vector 1. Small 2. Stable in the chosen host – usually E. coli 3. High copy number 4. Easy to purifiy 5. Can accommodate foriegn DNA 6. Single “cloning” sites 7. Selectable marker – antibiotic resistance 8. Easily introduced into host (transformation or transduction

Gene fusion systems – monitor the activity of a gene by fusing it to

Gene fusion systems – monitor the activity of a gene by fusing it to another Current favorites are the autofluorescent proteins He. La cells expressing gfp and rfp Clontech website

YAC(Yeast artificial chromsome) self-replicating vector that can be maintained in yeast Can accommodate large

YAC(Yeast artificial chromsome) self-replicating vector that can be maintained in yeast Can accommodate large insert Reeves et al. , 1992, Methods Enzymol. 216: 584 -603

BAC (bacterial artificial chromsomes) Derived from the F plasmid of E. coli low copy

BAC (bacterial artificial chromsomes) Derived from the F plasmid of E. coli low copy number (1 -2 copies per cell) Shizuya et al, 1992, PNAS 89: 9794 -8797

Informatics for Molecular Cloning n n Restriction Enzyme Site Analysis PCR Cloning –primer design

Informatics for Molecular Cloning n n Restriction Enzyme Site Analysis PCR Cloning –primer design Codon Usage Analysis Plasmid Construct – plasmid drawing

PCR primer selection n n n Primer Length Melting Temperature (Tm) Specificity Complementary Primer

PCR primer selection n n n Primer Length Melting Temperature (Tm) Specificity Complementary Primer Sequences G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches 3’-end Sequence

Primer 3

Primer 3

Codon Usage – differ from organisms

Codon Usage – differ from organisms

BASICS OF MOLECULAR CLONING Defining Cloning Cloning isBASICS OF MOLECULAR CLONING Defining Cloning Cloning is
Cloning Vectors Gene Cloning Cloning a definition FromCloning Vectors Gene Cloning Cloning a definition From
Cloning into Plasmids Restriction Fragment Cloning PCR CloningCloning into Plasmids Restriction Fragment Cloning PCR Cloning
CLONING TECHNIQUE What is cloning Cloning is aCLONING TECHNIQUE What is cloning Cloning is a
Unit Eight Cloning What is cloning Cloning isUnit Eight Cloning What is cloning Cloning is
Cloning 3 Types of Cloning Transgenic gene cloningCloning 3 Types of Cloning Transgenic gene cloning
Unit Eight Cloning What is cloning Cloning isUnit Eight Cloning What is cloning Cloning is
Cloning Wishful Thinking Cloning in Plants Plant CloningCloning Wishful Thinking Cloning in Plants Plant Cloning
Cloning Wishful Thinking Cloning in Plants Plant CloningCloning Wishful Thinking Cloning in Plants Plant Cloning
Cloning By Gina Patronaggio What is cloning CloningCloning By Gina Patronaggio What is cloning Cloning
Cloning Reproductive cloning vs Therapeutic cloning What isCloning Reproductive cloning vs Therapeutic cloning What is
Ethics of Cloning Types of Cloning Cloning andEthics of Cloning Types of Cloning Cloning and
Cloning into Plasmids Restriction Fragment Cloning PCR CloningCloning into Plasmids Restriction Fragment Cloning PCR Cloning
CLONING Sun Hwa Dong Cloning w Cloning producingCLONING Sun Hwa Dong Cloning w Cloning producing
Cloning What is cloning Cloning is a processCloning What is cloning Cloning is a process
Cheminformatics David Shiuan Department of Life Science andCheminformatics David Shiuan Department of Life Science and
Homology Modeling David Shiuan Department of Life ScienceHomology Modeling David Shiuan Department of Life Science
Protein Database David Shiuan Department of Life ScienceProtein Database David Shiuan Department of Life Science
Protein Targeting and Degradation David Shiuan Department ofProtein Targeting and Degradation David Shiuan Department of
Patent and Intellectual Property David Shiuan Department ofPatent and Intellectual Property David Shiuan Department of
Bioinformatics and Web Resources David Shiuan Department ofBioinformatics and Web Resources David Shiuan Department of
Protein Transduction David Shiuan Department of Life ScienceProtein Transduction David Shiuan Department of Life Science
Regulation of Gene Expression David Shiuan Department ofRegulation of Gene Expression David Shiuan Department of
Protein Folding David Shiuan Department of Life ScienceProtein Folding David Shiuan Department of Life Science