plasmid extraction.docx

To avoid shearing of the high-molecular-mass DNA, the cells were mixed with equal volumes of low-melting-point agarose 2% (w/v) in H2O at 45 C. The agarose-cell mixture was poured into moulds of approximately 1*2*4 mm in size, and allowed to solidify. The agarose plugs were incubated for 1.512 h at 37 C with 0.5 mg lysozyme m-1, 0.6 mg sodium N-lauroylsarcosine ml-1 in EET, followed by an overnight incubation at 55 C with 0.1 mg proteinase K ml-1, 10 mg SDS ml-1 in EET. The plugs were rinsed three times with TE10m1, pH 8 (10 mM Tris/HCl, 1 mMEDTA) and stored in ES buffer 0.5 M EDTA (pH 8.0), sodium N-lauroylsarcosine at 4C until use.general, a constant pulse time duration of 80 s was maintained during the total run time (24 h; 6 V cm21). For restriction analysis, a linear increasing pulse time from 1 to 25 s during the total run time (24 h; 6 V cm21) was used. At a later point an additional step of increasing pulse time from 25 to 30 s over 6 h was performed.The resuspended cell solution (1 ml) was warmed (45C, 3-4 min) and embedded into 1 ml of 1.2% low-melting temperature agarose containing 25% sucrose (45C). After solidifying, the plugs were removed from the molds, agitated in Cl/Tris/EDTA solution (200 mM NaCl, 10 mM Tris-Cl at pH 7.2, 100 mM EDTA at pH 8.0), removed from the NaCl/Tris/EDTA solution, agitated with a bacterial cell lysis solution followed by a proteinase K solution, and then cut and placed into a 1% agarose gel. DNA was separated using a clamped homogeneous electric field system (CHEF DR-III; Bio-Rad) at 1-50 sec linear ramp, 6 V/cm, 14C in 0.5X TBE buffer for 18 h. Concatamers of DNA ( ladder PFG marker, New England Biolabs) were used as molecular markers. To determine if the plasmids were linear or circular, the pulse times were changed: one gel was run at initial and final switch times of 30 sec; a second gel was run at initial and final switch times of 90 sec (31).The cells of R. opacus strain SAO101 (2.3107 to 5.3107) were embedded in a gel plug made of 1.0% (wt/vol). CleanCut agarose (Bio-Rad) and treated with enzymes by using a CHEF genomic DNA plug kit (Bio-Rad). Plugged samples were placed in slots of running gel made of 1.0% (wt/vol) pulsed-field certified agarose (Bio-Rad) and fixed by pouring molten 1.0% CleanCut agarose in the slots. Pulsed-field gel electrophoresis (PFGE) was performed with CHEF model DR III apparatus (Bio-Rad) under the following conditions: electrophoresis buffer, 0.5 TrisBorateEDTA; voltage, 200 V (6 V/cm); reorientation angle, 120; pulse time, linearly increased from 60 to 90 s; run times, 12 h; temperature, 15 C.Preparation and detection of linear plasmid DNA. Rhodococcus strains were grown in 10 ml of diluted LB medium to an optical density at 600 nm of 0.8. Cells were harvested by centrifugation at 2,000 g for 10 min, washed twice with 0.4 M sucrose, and recentrifuged. Then they were resuspended in 0.5 ml of TES (0.3 M sucrose, 0.25 M EDTA, 0.25 M Tris-HCl; pH 8) and mixed with 1.4% low-melting-point SeaPlaque agarose (FMC, Rockland, Maine) in TBE (0.49 M Tris, 0.49 M boric acid, 0.001 M EDTA; pH 8) (12). The resulting mixture was pipetted into plug molds (Bio-Rad Laboratories, Richmond, Calif.). After incubation at 4C for 15 min, the agarose plugs were pushed out of the molds into 5 ml of a lysozyme solution (1 mg/ml) in TES. After incubation at 37C for 2 h with swaying, the plugs were transferred to 5 ml of NDS (0.5 M EDTA, 0.01 M Tris pH 9, 1% wt/vol lauroyl sarcosine) containing proteinase K (1 g/ml) and incubated at 50C for 20 to 40 h. The proteinase K solution was predigested at 50C for 1 h. For preparation of non-proteinase K-treated plasmid DNA, proteinase K was omitted. For pulsed-field gel electrophoresis (PFGE), the plugs were inserted into the wells of agarose gels consisting of 1% agarose in TBE. Electrophoresis was performed with TBE as the running buffer at 14C by using a CHEF DRII PFGE system (Bio-Rad). The voltage, pulse time, and total running time were varied according to the size range of the fragments to be separated. Specific conditions are provided in the legends to the figures. Saccharomyces cerevisiae YNN295 chromosomes (Bio-Rad), a bacteriophage lambda ladder (Bio-Rad), and the Kb DNA ladder (Stratagene, La Jolla, Calif.) were used as size markers. Sodium dodecyl sulfat