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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
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Dideoxy DNA Sequencing
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Output from a DNA sequencing run
ds
dsDNA melting and primer annealing
Polymerase Chain Reaction (PCR)also uses DNA polymerase
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3’ 5’
5’ 3’
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Restriction Enzyme - EcoRI
The homodimeric enzymeEcoRI sits symmetricallyin the major groove of dsDNAand targets the palindromicsite GAATTC
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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
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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