BbsI NEB#R0539

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Set up a BbsI digest by mixing in an Eppendorf tube 300 μg plasmid DNA (see Note 9), 30 μL BbsI (150 U), and 60 μL 10× NEB2, and add H2O to 600 μL total volume. Mix well (without vortexing) and incubate at 37 °C overnight in a Thermomixer.

Protocol tips
Set up a BbsI digest by mixing in an Eppendorf tube 300 μg plasmid DNA (see Note 9), 30 μL BbsI (150 U), and 60 μL 10× NEB2, and add H2O to 600 μL total volume. Mix well (without vortexing) and incubate at 37 °C overnight in a Thermomixer.

Publication protocol

"BbsI Digestion of the Plasmid DNA
Set up a BbsI digest by mixing in an Eppendorf tube 300 μg plasmid DNA (see Note 9), 30 μL BbsI (150 U), and 60 μL 10× NEB2, and add H2O to 600 μL total volume. Mix well (without vortexing) and incubate at 37 °C overnight in a Thermomixer.

Check BbsI digestion on an agarose gel. Prepare a 0.8 % mini agarose gel in 1×TAE; add ethidium bromide to a final concentration of 0.5 μg/mL and allow to polymerize completely. Mix 0.5 μL of the BbsI digestion mix (250 ng DNA) with 4.5 μL H2O and 1 μL 6× gel loading dye. Load digested sample on the agarose gel alongside a sample of the undigested plasmid (250 ng) and 1 kb DNA ladder. Run the gel at 5 V/cm until the bromophenol blue and xylene cyanol are separated by about 2 cm and take a photograph. The BbsI-digested plasmid should be linearized (Fig. 3a) (see Note 10).Check by restriction analysis whether the BbsI digest was complete (see Note 11).

Place 2 μL of the BbsI digest mix (1 μg DNA) into each of the two Eppendorf tubes. Mix the first sample with 0.5 μL EarI (10 U), 1.8 μL 10× NEBuffer 2, and 15.7 μL H2O. Mix the second sample with 0.5 μL BamHI (10 U), 1.8 μL 10× NEBuffer 2, 2 μL 10× BSA, and 13.7 μL H2O. Incubate both tubes for 2 h at 37 °C.

Prepare a 12 % non-denaturing polyacrylamide gel using the BioRad Miniprotean 3 gel casting system. For a minigel with 1.5 mm spacers, prepare 10 mL of polyacrylamide gel: 3 mL 40 % Acrylamide (Acrylamide/Bisacrylamide 29:1), 2 mL 5×TBE, 4.93 mL H2O, 70 μL 10 % Ammonium persulfate, and 3.5 μL TEMED. Pour the gel and insert a comb with ten slots.

Add 4 μL 6× gel-loading buffer (without xylene cyanol) to the EarI and BamHI digests and load on the gel alongside the 100 bp DNA marker. Load 1× gel loading dye (with both marker dyes) in one slot to estimate the migration of the DNA fragments during the run (see Note 12). Run the gel in 1×TBE buffer at 1–8 V/cm until the bromophenol blue runs out of the gel.

Perform ethidium bromide post-staining by submerging the gel in 1×TBE containing 0.5 μg/mL ethidium bromide for 30–45 min shaking at room temperature. Wrap the gel in Saran Wrap and photograph with an ultraviolet transilluminator (see Note 13).

Only the expected fragments of 69 and 73 base pairs should be detected (Fig. 3b). If any residual fragments containing 24 extra base pairs are detected, add 1 μL BbsI (in 20 μL 1× NEB2) and continue the digest until completion (check again by EarI and BamHI restriction analysis). Do not continue to the next step until complete BbsI digestion is ensured (see Note 14)."

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