Maxplax lambda packaging extract

High-molecular-weight (HMW) DNA was isolated from 7-day cultured mycelia using the CTAB DNA isolation method. Genomic DNA was randomly sheared to approximately 40 kb in size. Then, the sheared molecules were size-selected using Pulse Field Gel Electrophoresis (Bio-Rad, Hercules, CA). The sheared DNA was ligated into the pCC2FOS vector (Epicentre, Madison, WI) after end-repair. The ligations were packaged using MaxPlax Lambda Packaging Extracts (Epicentre). The lambda phages carrying foreign DNA were used to infect EPI300-T1R E. coli cells (Epicentre). Positive fosmid clones were selected using lysogeny broth (LB)-chloramphenicol (12.5 μg/ml) plates. A total of 11,232 clones were selected and transferred to 96-well plates containing frozen LB media and stored at −80 °C.

To package into the λTriplEx2 vector, 25 µL of MaxPlax Lambda Packaging Extracts (Epicentre, Madison, WI, USA) was added into above ligation mixture, the detailed packaging procedure followed manufacturer’s instructions.

Activated sludge was collected from the aeration tank of a wastewater treatment facility in Japan. The sample was immediately stored at −80°C and simultaneously subjected to metagenomic DNA isolation. DNA was extracted from the sludge as previously described using a sodium dodecyl sulfate and proteinase K treatment (20 (link)). DNA was further purified for cloning into a fosmid following the methods of Rondon et al. (35 (link)). The size of extracted DNA was examined by agarose gel electrophoresis. Metagenomic libraries using DNA extracted from the activated sludge was constructed using the commercial fosmid vector, Copy Control™ pCC1Fos (Epicentre). The library was constructed by DNA size fractionation, a clean-up of metagenomic DNAs, and subsequent ligation into a fosmid vector. The ligation mixture was then packaged into lambda phages using MaxPlax Lambda Packaging Extracts (Epicentre). The packaged library was transfected into E. coli EPI300. E. coli transformants were selected on LB agar supplemented with chloramphenicol. The presence of recombinant plasmids and polymorphisms in insert DNA were examined by agarose gel electrophoresis of the EcoRI digestion of purified plasmids from randomly selected E. coli transformants.

The genomic DNA was prepared via the Hoffman and Winston method [51 (link)]. The DNA was then sheared by repeated aspiration and expulsion of the samples from the pipette tip (50–100 times). The DNA fragments were then isolated for a range of 25–50 kb via electrophoresis in a 20 cm long, 1% agarose gel at 30 V for 16 hours. The DNA products were end-repaired with the end-repair enzyme mixtures included in the CopyControl™ Fosmid Libary Production kit (EPICENTRE: Madison, Winconsin) and then ligated into fosmid vector PCC1FOS according to manufacturer's protocol. Packaging of the fosmid clones were done using MaxPlax Lambda Packaging Extracts (EPICENTRE: Madison, Winconsin). Packaged fosmids were stored at 4°C with the addition of chloroform and phage dilution buffer (10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 10 mM MgCl₂). Serial dilutions of packaged fosmid clones (phage particles) were done to calculate the clone titer. Phage particles were mixed with EPI-T1 cells in a ratio of 100 μL cells for every 10 μL of diluted phage particles. These mixtures were then incubated at 37°C for 20 min. Infected bacteria were then spread on LB plate + 12.5 μg/mL chloramphenicol and incubated at 37°C overnight to select for fosmid clones. Induction of fosmid clones were then carried out according to the manufacturer's protocol using 1000X CopyControl induction solution.

Genomic DNA was collected from a T. mesophilum cell pellet (following cultivation for 4 days at 30 °C in sea water containing 5 g/L yeast extract and 10 g/L tryptone) using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The resulting genomic DNA was size separated by agarose gel electrophoresis (1% low melting point agarose gel, 30 V for 15 h). The DNA above 23 kb was recovered from the gel by digestion with a thermostable β-agarase (Nippon Gene, Tokyo, Japan). Purified DNA was blunt-ended using the End-It DNA End-Repair Kit (Epicentre, Madison, WI, USA), followed by ligation into pCC1FOS fosmid vector (Epicentre). This mixture was subjected to packaging with the MaxPlax Lambda Packaging Extract (Epicentre), and then transfected into Escherichia coli EPI300-T1R (Epicentre) according to the manufacturer’s protocol. Transformants were plated to Luria-Brrtani (LB) agar (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 10 g agar per L of deionized water) containing chloramphenicol (30 μg/mL) to yield a T. mesophilum genomic library comprising a total of 9.6 × 10⁴ clones with an average insert length of 35 kb.

S. somaliensis SCSIO ZH66 genomic DNA was partially digested with Sau3AI, and fragments with the size of 40-50 kb were recovered and dephosphorylated with CIAP, and then ligated into SuperCos1 that was pretreated with XbaI, dephosphorylated, and digested with BamHI. The ligation product was packaged into lambda particles with the MaxPlax Lambda Packaging Extract (Epicenter, Madison, WI, USA) as per the manufacture’s instruction and plated on E. coli Top10. The titer of the primary library was about 2 × 10⁶ cfu per μg of DNA. Specific primers were designed per the draft genome sequence for library screening against 2500 colonies by PCR (Additional file 1: Table S3).