FAQ: What are the design rules for the target-specific oligos?

A 20 nucleotide target-specific sequence is selected.  We recommend using a target DNA selection program.  Make sure to remove the PAM (NGG) sequence.  First, the T7 promoter sequence (5´ TTCTAATACGACTCACTATA 3´) is appended to the 5´ end of the target-specific sequence.  If the first nucleotide of the target specific region is a G, no nucleotides need to be added.  If there is no G present, add one G to the 3´ end of the T7 promoter sequence (..TATAG..), just upstream of the target-specific sequence.  Next, append the 14-nucleotide overlap (5´ GTTTTAGAGCTAGA 3´) to the 3´ end of the target-specific sequence.  The complete sequence represents the target-specific oligo to be ordered.  This oligo will hybridize with the complementary sequence on the scaffold oligo provided in the 2X sgRNA Reaction Mix and create the template for dsDNA synthesis by the DNA polymerase contained within the sgRNA Enzyme Mix.