Noti hf neb9/3/2023 ![]() These engineered enzymes have the same specificity as the native enzyme, with the added benefit of reduced star activity, rapid digestion (5-15 minutes) and 100 activity in CutSmart ® Buffer. Note that EcoRI and SspI have HF versions ( EcoRI-HF and SspI-HF) which are supplied with rCutSmart Buffer. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF ®) Restriction Enzymes. In most cases, DpnII requires a sequential digest. 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. NEB currently supplies three enzymes with unique buffers: EcoRI, SspI and DpnII. DNA was then purified using 45 μL SPRI beads. Thermo Scientific NotI restriction enzyme recognizes GCGGCCGC sites and cuts best at 37C in O buffer (Isoschizomers: CciNI). 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. 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. NotI-HF restriction enzyme site in pSB1C3 from NEBcutter V2.0. Phi X 174 nanopore libraries were constructed using a shotgun-ligation approach. IGEM:Hong Kong HKUST/Investigations/Performance of NotI-HF in 5, 15 and 30 minutes.
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