SspI NEB#R0132

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	0.5 to 4 μg of Phi X 174 gDNA (Thermo Scientific) was restriction-digested using 5U of SspI (NEB) in 1× SspI Reaction Buffer for 2 hours at 37°C to linearize DNA and produce blunt ends followed by SPRI bead purification.

Protocol tips
0.5 to 4 μg of Phi X 174 gDNA (Thermo Scientific) was restriction-digested using 5U of SspI (NEB) in 1× SspI Reaction Buffer for 2 hours at 37°C to linearize DNA and produce blunt ends followed by SPRI bead purification.

Publication protocol

Phi X 174 nanopore libraries were constructed using a shotgun-ligation approach. 0.5 to 4 μg of Phi X 174 gDNA (Thermo Scientific) was restriction-digested using 5U of SspI (NEB) in 1× SspI Reaction Buffer for 2 hours at 37°C to linearize DNA and produce blunt ends followed by SPRI bead purification. DNA was resuspended in 42 μL Elution Buffer (EB, QIAGEN) followed by addition of 5 μL 10× T4 DNA Ligase Buffer (NEB), 1 μL of each annealed adaptor (FT-½NotI and HP-½NotI), and 1 μL (400U) of T4 DNA Ligase (NEB) and incubated overnight at 16°C. Ligase was heat-inactivated at 65°C for 15 minutes followed by cooling on ice and addition of 1 μL 10× T4 DNA Ligase Buffer, 1 μL (1U) USER Enzyme (NEB), 1 μL (20U) NotI-HF (NEB), and 2 μL (10U) λ Exonuclease (NEB) and incubation at 37°C for 2 hours. DNA was then purified using 45 μL SPRI beads. A subset of samples were gel-size-selected to remove adaptor-dimer bands on a 1% agarose gel (SeaKem) and purified using the column-based Gel Purification Kit (QIAGEN) and eluted in EB.

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