Biochemistry 2280A Lecture Notes - Lecture 6: Ecori, Bamhi, Sticky And Blunt Ends
5 Mar 2014
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Hybridization of primers ( 60 degrees celsius); dntps are here too, don"t mind. In presence of dntp, extend primers of both strands (72 c) One cycle gets you two dna molecules from one. Pcr is repeated, two molecules denature, anneal primers + extend; after cycle 2 4. 3rd cycle 8 molecules; start to see molecules that begin and end with primer sequences. # of copies after n cycles is 2^n; 2^30 ~ 1 billion. Discovery of thermostable dna polymerase (taq polymerase; isolated from dna of hot. Thermocyclers that oscillate between the 3 required temperatures (50c, 94c, 70c) Run through 3 temperatures automatically; takes 1. 5 hrs to do 30 cycles now. Exponential amplification of any dna from a source in which it is found as little as once. Required reagents for pcr: template dna, 2 oligonucleotide primers which flank tfg, dntps, dna polymerase. Dna is homogenous; charge to mass ratio is similar, differ in length separate.
