Topo shotgun subcloning kit

Chromosomal DNA from S. carnosus was isolated [57 (link)] using lysostaphin (0.5 mg/mL) for cell lysis. Genomic DNA was sheared using the nebulizer from the TOPO Shotgun Subcloning Kit (Invitrogen, Carlsbad, CA, USA) and separated on a preparative agarose gel. Five pools of DNA from 1.7 to 3 kbp were isolated, cleaned and treated with Klenow fragment to generate blunt ends. DNA from each pool was used for ligation into pKT25 [65 (link)], which had been cut with SmaI and dephosphorylated by rAPid Alkaline Phosphatase (Roche, Mannheim, Germany). The resulting plasmids were transformed into NEB 5-alpha electrocompetent E. coli cells (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Ligation procedure was performed twice, resulting in ten transformations. Each transformation was plated on three LB/Kanamycin (50 µg/mL) plates (15 cm in diameter) and incubated overnight at 37 °C. All colonies from the same pool were combined and stored in 50% glycerol at −80 °C. From each pool, an aliquot was used to inoculate LB media for plasmid isolation (“prey” vectors). Over 80% of the plasmids contained genomic DNA inserts, and cell wall related genes like pbpA, pbpF, pbp4, rodA and murJ could each be amplified in at least one subpool. SCA_1084 (pbp2) was cloned into pUT18C [65 (link)] as “bait” vector.

BAC DNA was mechanically sheared either with nebulizers (for 30 sec) in the TOPO shotgun sub-cloning kit (Invitrogen), or with sonication. Blunt-end fragments were subcloned into pCR4, pCRII (Invitrogen), or pUC118 (Takara Bio). Shearing with sonication, and pUC118 subcloning, were performed by the Kazusa DNA Research Institute. Shotgun subclones were sequenced from both ends by the Research Resource Center (BSI, RIKEN), or the Kazusa DNA Research Institute. Raw sequence data were base-called, vector-trimmed for each, end-clipped to remove low-quality regions, and assembled by CodonCode Aligner (CodonCode: http://www.codoncode.com/aligner/). Assembled contigs were queried against C. reinhardtii V4 protein models, or V. carteri protein models (Ver. 2, JGI and male and female MT, NCBI) using BLASTX to identify protein coding genes (Merchant et al. 2007 (link); Ferris et al. 2010 (link)).

The Northern pike fosmid genomic DNA library was constructed by Bio S&T (Québec, Canada) from high molecular weight DNA extracted from the liver of a male E. lucius from Ille et Vilaine, France, using the CopyControl Fosmid Library Production Kit with pCC1FOS vectors (Epicentre, USA) following the manufacturer’s instructions. The resulting fosmid library contained around 500,000 non-amplified clones that were arrayed in pools of 250 individual fosmids in ten 96-well plates.
Northern pike fosmid clones were screened by PCR (S5 Table) to identify individual fosmids containing amhby and amha. To sequence the two ~ 40 kb fosmids, purified fosmid DNA was first fragmented into approximately 1.5 kb fragments using the Nebulizer kit supplied in the TOPO shotgun Subcloning kit (Invitrogen, Carlsbad, CA), and then sub-cloned into pCR4Blunt-TOPO vectors. Individual plasmid DNAs were sequenced from both ends with Sanger sequencing using M13R and M13F primers. The resulting sequences were then assembled using ChromasPro version 2.1.6 (Technelysium Pty Ltd, South Brisbane, Australia).