SmiI (SwaI) restriction enzyme

Protocol tips

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	To estimate the genome size, DNA was digested with two rare-cutting restriction enzymes, PmeI (NEB, Ipswich, MA) and SmiI (recognition site identical to that of SwaI) (TaKaRa).

Protocol tips
To estimate the genome size, DNA was digested with two rare-cutting restriction enzymes, PmeI (NEB, Ipswich, MA) and SmiI (recognition site identical to that of SwaI) (TaKaRa).

Publication protocol

Preparation and restriction digestion of intact genomic DNA in agarose plugs were performed according to the protocol reported previously (20). To detect plasmids, DNA was digested with S1 nuclease (TaKaRa, Tokyo, Japan). To estimate the genome size, DNA was digested with two rare-cutting restriction enzymes, PmeI (NEB, Ipswich, MA) and SmiI (recognition site identical to that of SwaI) (TaKaRa). Pulsed-field gel electrophoresis (PFGE) was carried out in a CHEF-DRII gel electrophoresis system (Bio-Rad, Richmond, CA) in 0.5 Tris-borate-EDTA (TBE) buffer at 14°C in a temperature-controlled cooling unit. To separate DNA fragments of 0.1 to 2 Mb, contour-clamped homogeneous electric field (CHEF) electrophoresis was conducted in a 1% pulsed-field agarose gel (pulse time, 10 to 120 s; field strength, 6 V cm1 ; angle, 120°; run time, 24 h). DNA fragments of 1 to 3 Mb were separated in a 0.8% pulsed-field agarose gel (pulse time, 500 s; field strength, 3 V cm1 ; angle, 106°; run time, 48 h). DNA fragments of 3 Mb were separated in a 0.8% pulsedfield agarose gel (pulse time, 1,800 s; field strength, 2 V cm1 ; angle, 106°; run time, 72 h). Chromosomes of Saccharomyces cerevisiae (0.1 to 2 Mb and 1 to 3 Mb), Schizosaccharomyces pombe (3 Mb), and Hansenula wingei (3 Mb) (Bio-Rad) and the lambda DNA ladder (0.1 to 2 Mb) (TaKaRa) were used as size standards.

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