**2.2 BAC purification and evaluation**

In order to obtain sufficient BAC DNA for PCR based clone selections, pulse field gel electrophoresis (PFGE) mediated evaluations and/or direct sequencing, around 3~6 ml liquid culture was inoculated for each colony. Prior to the purification of the BAC DNA, 400 l of the saturated culture was mixed with 80 l of 40% glycerol solution and stored at -80C. The general protocol to purify the plasmid DNA was modified for the BAC DNA as follows: a bacterial cell pellet from the saturated culture (up to 3 ml) was collected into a 1.5 ml tube by serial centrifugations, and was suspended into 100 l of solution I. The suspension was gently lysed by adding 200 l of freshly prepared solution II (slowly inverting the tube four times so as to make the suspension nearly transparent) and carefully neutralised by adding 150 l of cold solution III (gently inverting the tube twice to mix the contents evenly). After centrifugation at 15,000 rpm for 10 min at 4C, the supernatant was overlaid on 500 l of phenol chloroform mixture, and the content was evenly blended by gently shaking the tube three times. After centrifugation at 15,000 rpm for 5 min at room temperature, the upperlayer was transferred to a 1.5 ml tube filled with 350 l of isopropanol, mixed and reserved at -30C for at least 30 min. After centrifugation at 15,000 rpm for 10 min at 4C, the pellet including the BACs and RNA was washed with 75% ethanol and dissolved into 30 l of distilled water (DW) containing RNase at 60C for 10min. 7.5 l of the BAC solution can be used for visualising the DNA fragment on normal 0.6% agarose/TAE gel electrophoresis or 1% agarose/TBE PFGE. For sequencing, polyethyleneglycol-based precipitation was carried out overnight at 4C, and the pellet was dissolved into DW to measure the optical density at OD260 by NanoDrop (Thermo Fisher Scientific).

To obtain good amounts of BAC DNA with high quality for trans-genesis, a CsCl density gradient ultracentrifugation-based purification method or a purification kit (NucleoBond BAC 100, Macherey-Nagel) were utilised. For CsCl purification, 1,500 ml LB was inoculated for each clone from the glycerol stock. For the kit, 250 ml of the culture was harvested and BAC DNA was purified as the manufacture's protocols recommended.
