The
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
