Pfge agarose

BAC DNA was isolated by standard alkaline lysis from 5 ml liquid cultures. Subsequently, the integrity of BACmid DNA was analyzed by digestions with restriction enzyme XhoI and separation in 0.8% PFGE agarose (Bio-Rad) gels and 0.5×TBE buffer by pulse field gel electrophoresis at 6 V/cm, 120 degree field angle, switch time linearly ramped from 1 s to 5 s over 16 h (CHEF DR III, Bio-Rad). For characterization of insertion or deletion of the aphaI selection marker, recombinant BACmids were analyzed by PCR, and the recombined regions within the BAC DNA were controlled by sequence analysis (ABI 3130XL genetic analyzer, Weiterstadt, Germany) in order to confirm the correct deletion sequences and to exclude accidental mutations. Furthermore, the integrity of the complete viral genome of representative Bac16 recombinants was confirmed by next-generation sequencing using Nextera DNA Sample Preparation system and the MiSeq Reagent Kit, 300 Cycles on the Illumina MiSeq system. NGS data were analyzed by Genome Workbench 5 (CLCbio, Aarhus, DK).

PFGE was performed as previously described^{63 (link),73 (link)}. Briefly agarose plugs have been run in a 1% agarose gel (Bio-Rad PFGE agarose) prepared in 150 ml of TBE 0.5× and the migration chamber was filled with 3 litres of TBE 0.5×. The Amersham Gene Navigator System was used with a water refrigerating bath set at 4 °C. The gel was run at 165 V for 24 h with 30 s pulses (step wise) for the separation of the chromosomes XII.
For the isolation of the rDNA array genomic DNA embedded in the plugs was digested with BamHI^{74 (link)} and digested plugs were run into the PFGE as described above using the following run conditions :100 V for 69 h (step 1 = 68 h with pulses of 300 s and step 2 = 1 h with 900 s pulses; the two steps were connected by the interpolation mode). During the PFGE run pulses went from 300 to 900 s.