PP10 - Polymerase Chain Reaction (PCR) Extraction of...

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Extraction of Plasmid DNA Polymerase Chain Reaction (PCR)
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What You Will Learn  from This Class-- Principles  of DNA Replication Polymerase Chain Reaction  to Amplify Large Quantities of  DNA in a Few Hours The   Alkaline Lysis   Mini-Prep Procedure to Isolate  Plasmid   DNA from  Bacterial  Cells
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Polymerase Chain Reaction (PCR)
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DNA Replication
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What you need during DNA replication: -  DNA-dependent DNA polymerase -  dNTPs -  a template DNA -  a primer strand with a 3’-OH end
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Enzyme involved in DNA replication DNA-dependent DNA polymerase  synthesize DNA from dNTPs   requires a template strand requires a primer strand with a 3’-OH end  all synthesize from 5’ to 3’ (add nt to 3’ end  only)
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Mo vie  – DNA po lym e riza tio n http://www.sbs.utexas.edu/huq/bio344/
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5' 3' 3' 5' 5' 5' DNA Polymerase III acts   here DNA Polymerase I extends one Okazaki fragment and removes the RNA from another. DNA Ligase then joins fragments   together. ssDNA binding protein  ( SSB ) Helicase (DnaB) Primase 3' Gyrase S p i n n i n g a t 1 0,00 0 r p m A   p ro k a ry o ti c   fo rk  i s  tra v ellin g   a t  5 0  to  1 0 0  k b   /   m in u te. Eu k a ry o ti c   fo rk s   tra v el  a t  0.5  -  5  k b   /   m in u te. Primosome DNA Replication in a living cell Replisome
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What you need for PCR 1. DNA Template 2.  Two Primers 3.  Taq DNA Polymerase 4.  dNTPs 5.  Appropriate buffers and cofactors
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Mo vie  – DNA po lym e riza tio n http://www.sbs.utexas.edu/huq/bio344/
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Application of PCR 1. C lo ning  a  pa rtic ula r fra g m e nt o f DNA.  2.  S e q ue nc ing  o f DNA 3. Mo dific a tio n o f PC R pro duc ts  –intro duc tio n o f re s tric tio n s ite s , pro m o te rs ,  rib o s o m e   b inding   s ite s ,  s ite -dire c te d  m uta g e ne s is e tc . 4.  Re ve rs e   tra ns c riptio n  PC R  (RT  PC R),  c o ns truc tio n  o f  c DNA lib ra ry, diffe re ntia l dis pla y PC R 5. Q ua ntita tive  RT-PC R (q RT-PC R) 6.  G e no m e   m a pping   -  uniq ue   re g io ns   de te c te d  in  la rg e g e no m e s , C hro m o s o m a l m a pping
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7. DNA fingerprinting and Genetic analysis. 
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PP10 - Polymerase Chain Reaction (PCR) Extraction of...

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