530L_notes_lecture3 - V. PCR: the polymerse chain reaction...

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V. PCR: the polymerse chain reaction A. requirements 1. template DNA Gene, DNA region, anything we’re interested in making a lot of copies of… Variety of different forms: As big as total genomic dna from ecoli, or plasmic dna, DNA from tissue samples, blood…etc Any type of source of DNA…can use PCR. 2. Primers (ssDNA; DNA oligonucleotides) Need pair of primers: two single stranded poly nucleotide primers since DNA is antiparallel. 18-35 nucleotides in length Chemically synthesized 3. Thermostable DNA polymerase Make copies of DNA. Thermal stable DNA polymerase. Common: TAQ – thermos aquaticus – lives in hot springs…80-90 degree C. Very very thermal stable. Common: vent polymerase – from species of bacteria that lives in hydrothermal vent. Both incredibly stable. 4. dNTPs (substrate for DNA polymerase; dATP,dCTP,dGTP,dTTP) Deoxy nucleoside tri phosphate Remove a nucleotide and attach Takes place in vitro. 5. Reaction buffer TAQ purchase comes with reaction buffer. Vent DNA polymerase also comes with buffer.
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Depending on the enzyme, look out for whether or not it contains Mg – essential component for nucleotides to attach. TAG reaction buffer does not come with Mg; however, Vent does. 20mM concentration. Need to dilute to 2mM (magic #). 6. Thermocycler (PCR Machine) Small machine whose job is to get really hot and really cold over and over again. B.
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This note was uploaded on 03/14/2011 for the course CHEM 530L taught by Professor Hogan during the Spring '09 term at UNC.

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530L_notes_lecture3 - V. PCR: the polymerse chain reaction...

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