Home FAQs Will I get a higher yield if I allow my RCA step in the EnClose™ Cell-free dbDNA™ Synthesis (NEB #E9301) protocol go for more than 3 hours (e.g., overnight)?

FAQ: Will I get a higher yield if I allow my RCA step in the EnClose™ Cell-free dbDNA™ Synthesis (NEB #E9301) protocol go for more than 3 hours (e.g., overnight)?

Incubation beyond 3 hours does not typically increase overall RCA product yield using the recommended reaction conditions. If the incubation time is increased, there is a chance for more amplification of nonspecific products in the absence of template. This will increase the burden of enzymatic cleanup in the restriction enzyme/exonuclease step of the workflow that may lead to lower purity of the desired dbDNA product. If more yield is needed in the RCA reaction due to the characteristics of the plasmid, we recommend increasing the amount of input DNA and dNTPs, or setting up additional RCA reactions in parallel with the same input plasmid amounts.