Home FAQs Why does my RCA reaction appear cloudy when performing the EnClose™ Cell-free dbDNA™ Synthesis protocol (NEB #E9301)?

FAQ: Why does my RCA reaction appear cloudy when performing the EnClose™ Cell-free dbDNA™ Synthesis protocol (NEB #E9301)?

A cloudy RCA reaction is not necessarily cause for concern. During the RCA reaction, there is an accumulation of pyrophosphate generated from the incorporation of the dNTPs during amplification, which can make the reaction look opaque and cloudy. Although pyrophosphatase is included in the RCA reaction, the amount of pyrophosphate generated could surpass the ability of the enzyme to hydrolyze inorganic pyrophosphate, or the pyrophosphatase was accidentally excluded from the reaction. Either way, this should not greatly reduce the yields of the RCA reaction itself.