GenomiPhi first-strand cDNA - Can GenomiPhi be used on single-stranded DNA? (Jun/27/2008 )
I have cDNA that I made from RTing total RNA using poly-T primers. I am trying to amplify some genes from this cDNA (many genes from few taxa) and it is becoming apparent that I will have to clone all of them. Could I just GenomiPhi the cDNA?
from what I have understood of the amplification mechanism in the GenomiPhi reaction, I would say you cannot use cDNA because it is single stranded... but i might be wrong.
what is the source of your cDNA ? if you are going to clones several genes from different taxa out of it, I can only think of an environmental sample... tell us it is not bacteria.
The cDNA starts out as ssDNA, but after the first primer by random nonamers, it is dsDNA. I see no reason why it should not amplify. There may be issues with the statistical properties of the amplification.
Ph3no is trying to tell you, perhaps, that dT oligo amplification of bacterial mRNA will typically not work well, since most bacterial mRNA is not poly-adenylated. Random nonamers are typically used to prime prokaryotic mRNA reverse transcription reactions (or a gene specific primer).