Originally posted by pmiguel
View Post
Think about what you are doing! Remember each PCR cycle potentially doubles the amount of the initial template. So, say you are amplifying a 1 kb segment of a 1 billion bp genome. 1 ug of DNA from this organism will contain 1 pg of the segment of interest. How many PCR cycles, theoretically, do you need to obtain enough product?
10 cycles? 1 thousand-gold amplification (pg become ng)
20 cycles? 1 million-fold amplification (pg become ug).
30 cycles? 1 billion-fold amplification (pg become mg -- impossible because your PCR will run out of primers, nucleotides, etc.)
Also, the purpose of the (2nd) step-out PCR is to just add the rest of the adapters -- 3 or 4 cycles should be plenty!
Of course PCR can't achieve a doubling of template concentrations in each cycle -- but this gives you an idea of how heavy the hammer you are smashing your project with!
--
Phillip
Comment