Fig. 2

Optimization of long ssDNA production using T7 exonuclease and restriction enzymes. a Schematic of long ssDNA production using T7 exonuclease (one-step PCR method). b T7 exonuclease reaction on dsDNA amplified using three different combinations of primers (PS-modified (PS) or non-modified (noPS) for the forward and reverse primers). The DNA sequence of the donor for Cas12a-mediated mNG tagging of HNRNPA1 was used in this experiment. The bottom image is of the same gel as the top one, with higher brightness and contrast. ssDNA shows higher mobility than dsDNA of the same length. Asterisks show undigested dsDNA remnants. c Schematic of ssDNA production by two-step PCR and T7 exonuclease (T7 method). d “PS-PS” dsDNA was prepared with one-step or two-step PCR and subsequently subjected to the T7 exonuclease reaction. Plot profiles for each lane are shown below the gel electrophoresis images. The two images are cropped from the same gel image. e Production of long ssDNA donors using the one-step and the two-step PCR methods. The bottom image is of the same gel as the top one, with higher brightness and contrast. f Schematic of ssDNA production using T7 exonuclease and restriction enzymes (T7RE method). After two-step PCR and T7 exonuclease reaction, the indicated four restriction enzymes digest dsDNA remnants to produce short dsDNA fragments which can be further degraded by T7 exonuclease. g ssDNA production by the T7 and the T7RE methods. The DNA sequence (sense strand) of the donor for Cas9-mediated mNG tagging of HNRNPA1 was used in this experiment. The last two lanes contain column-purified DNA products of both reactions. Plot profiles for the last two lanes are shown below the gel electrophoresis image. The two images are cropped from the same gel image. Full-length gels are presented in Fig. S6