nebuffer1  (New England Biolabs)

Journal: Frontiers in Microbiology

Article Title: The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

doi: 10.3389/fmicb.2021.657356

Figure Lengend Snippet: Characterization of TkoII restriction endonuclease activities. (A) Predicted TkoII recognition sites in plasmid pBR322 are at positions 1093, 2944 and 4355. (B) TkoII was incubated with pBR322 in 1X NEBuffer 1 supplemented with SAM and 90mer trans-DNA for 30 min at 65°C. Reactions were halted with Proteinase K and loading dye and separated on a 1% agarose gel. Lane 1 is a 1 kb DNA ladder. Lane 2 is pBR322 alone and lanes 3–7 are decreasing amounts of TkoII (10–0.62 nM). (C) TkoII cleaves pUC19 at a single site. (D) pUC19/TkoII cut fragments were analyzed by run off sequencing to determine TkoII cut sites on the top and bottom strands: TTCAAG(N) 10 /(N) 8 .

Article Snippet: TkoI and TkoII REase activity was assayed by mixing TkoI (400 nM) or TkoII (10 nM) with plasmid pBR322 (1 μg) or pUC19 (3 μg), SAM (80 μM), 90mer trans-DNA (0.2 μM) in 1× NEBuffer 3 (100 mM NaCl, 50 mM Tris–HCl, pH 7.9, 10 mM MgCl2 ) for TkoI or NEBuffer 1 (10 mM Bis-Tris–Propane-HCl, pH 7, 10 mM MgCl2, 1 mM DTT) for TkoII in a 50 μL reaction and incubated at 65°C for 30 min. For pBR322 reactions, four two-fold serial dilutions were performed.

Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Sequencing