Hello everyone,

We are trying to set up a one-step PCR for COVID19. We are trying to implement the Midnight 1200bp, by using a one-step RT-PCR protocol instead of the recommended two-step alternative. We are using Takara's PrimeScript. We are able to get a product with ARTIC primers (standard protocol) ~400 bp PCR product, regardless of sample quality, however in Midnight Protocol when we use better samples, the results seem to be more reliable however there is still a large amount of variability compared to one another. Would anyone have an idea on how I can get rid of this problem? Should I try and do the PCR two-step?

Thanks for any help

Ege