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Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR). Bridges 2014. Polymerase Chain Reaction. Simple reaction Produces many copies of a specific fragment of DNA Live replication takes much longer Only requires a small amount of DNA ( ng ) Many types of PCR. What goes into a PCR?. Mostly water

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Polymerase Chain Reaction (PCR)

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  1. Polymerase Chain Reaction (PCR) Bridges 2014

  2. Polymerase Chain Reaction • Simple reaction • Produces many copies of a specific fragment of DNA • Live replication takes much longer • Only requires a small amount of DNA (ng) • Many types of PCR

  3. What goes into a PCR? • Mostly water • Autoclaved MilliQ • Forward Primer • Front end of target DNA sequence • Reverse Primer • Back end of target DNA sequence • Template DNA

  4. What goes into PCR? • PCR Buffer • Balances pH levels • MgCl2 • Helps target specificity and stabilizes DNA • dNTPs • Nucleotides (A, G, C, T) • Polymerase • Polymerase used to “build” target sequence

  5. PCR is SENSITIVE! • Need specific amounts of each reagent • Too much or too little can RUIN a PCR • Highly subject to contamination • Use sterile technique! • Needs specific thermocycling program

  6. Thermocycler

  7. Thermocycler program • Denature • Anneal • Extend

  8. Biological Response to Thermocycler

  9. Exponential growth of template DNA

  10. 16S rRNA • Ribosomal Sequence • Ribosomes make proteins • All bacteria have 16S • Has conserved and hypervariable regions • Conserved regions- primer binding site • Hypervaribale regions are for species identification

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