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. 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. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF ®) Restriction Enzymes. NotI-HF (and most other enzymes) is also active in 1× CutSmart Buffer at 37C. DNA was then purified using 45 μL SPRI beads. The following protocol is current for NEB enzymes at the time of this. 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☌. HF enzymes also exhibit dramatically reduced star activity. High Fidelity (HF) Restriction Enzymes have 100 activity in rCutSmart Buffer single-buffer simplicity means more straightforward and streamlined sample processing. 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 has a High Fidelity version NotI-HF ® ( NEB R3189 ). 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).
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