#Noti hf neb for free

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.įull paper Login or join for free to view the full paper. DNA was then purified using 45 μL SPRI beads. Ligase was heat-inactivated at 65☌ 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☌ for 2 hours. Thermo Scientific NotI restriction enzyme recognizes GCGGCCGC sites and cuts best at 37C in O buffer (Isoschizomers: CciNI). generate monomer units from the DNA concatemers, rolling cycle amplified DNA was digested with 100 U NotI-HF (NEB) for 16 h at 37C in a 1 ml reaction. 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☌. 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☌ to linearize DNA and produce blunt ends followed by SPRI bead purification. New England Biolabs single cutting restriction noti hf enzyme Single Cutting.

Phi X 174 nanopore libraries were constructed using a shotgun-ligation approach. All HF-restriction enzymes come with Gel Loading Dye, Purple (6X).

Noti hf neb