NEB scientists have screened a large collection of single-stranded DNA guides targeting randomized single- and double-stranded DNA sequences in order to provide to following useful tips to help with designing guides for your own unique applications. The underlying mechanisms of in situ prokaryotic argonaute guide production are an area of ongoing research, and there are a variety of factors that can affect the activity of guide/argonaute combinations that have yet to be elucidated. As such, the following recommendations represent a “best foot forward” approach for guide design, however they do not guarantee that a guide designed in accordance will be functional.
GUIDEline #1: Begin guides for TtAgo with a thymidine
GUIDEline #2: Avoid adenosine in the twelfth position of guides for TtAgo
GUIDEline #3: Select an optimal target sequence GC content for TtAgo
GUIDEline #4: ET SSB may aid in cutting higher GC content substrates under specific conditions
GUIDEline #5: ThermoPol® Reaction Buffer with 2mM magnesium(II) is optimal for most reactions
General Usage Notes: