2 step PCR for FACS based CRISPR screens

[Ashwin Iyer]

Dear genome engineering members, 

My question is pertaining to PCR1 + PCR2 with regard to limited sample size (from enrichment screens). 2 step PCR improves sensitivity no doubt, but when we do it for samples of low numbers, is there a key advantage over a 1 step PCR? Also when we do 2 step, we are also doing it on the pre-sort. So I am a bit confused how 2 step gets you better signal/noise for low abundance targets compared 1 step?

Could someone please explain to this concept to me?

Best,

Ashwin Iyer