melting of dsdna

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1 ds dsDNA melting and primer annealing Melting of dsDNA Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC) http://www.biosyn.com/Gizmo/Tools/Oligo/Oligonucleotide%20Properties%20Calculator.htm

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Page 1: Melting of dsDNA

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ds

dsDNA melting and primer annealing

Melting of dsDNA

Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)http://www.biosyn.com/Gizmo/Tools/Oligo/Oligonucleotide%20Properties%20Calculator.htm

Page 2: Melting of dsDNA

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Dideoxy DNA Sequencing

Page 3: Melting of dsDNA

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Output from a DNA sequencing run

ds

dsDNA melting and primer annealing

Polymerase Chain Reaction (PCR)also uses DNA polymerase

Page 4: Melting of dsDNA

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3’ 5’

5’ 3’

Page 5: Melting of dsDNA

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Restriction Enzyme - EcoRI

The homodimeric enzymeEcoRI sits symmetricallyin the major groove of dsDNAand targets the palindromicsite GAATTC

Page 6: Melting of dsDNA

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Page 7: Melting of dsDNA

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3’ 5’

5’ 3’

Add restrictionsite

PCR can be used to add a restriction site to DNA

Eukaryotic gene for insulin expressed in E. coli

Page 8: Melting of dsDNA

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Making cDNA from mRNA transcript

Overview of Prokaryotic Heterologous Over-Expression

Strong promoter – Plac

Ribosome binding –Shine-Dalgarno, AGGA / purine rich ~ 7 b.p. before start codon: ATG

Multi-cloning site to put your gene in with correct frame and direction.

Plasmid 3-6 kb

E. coli Genome 5,000 kb