title: Lactobacillus paracasei 7.8 K
status: Public on Dec 06 2006
contact.city: Lincoln
contact.first: Yong Jun
contact.last: Goh
contact.email: yjgoh@ncsu.edu
contact.state: NE
contact.address: 332 Food Industry Complex
contact.department: Food Science and Technology
contact.country: USA
contact.institute: University of Nebraska
organism: Lacticaseibacillus paracasei
manufacture_protocol: A shotgun genomic library of L. paracasei was constructed using Zero Blunt TOPO Cloning Kit (Life Technologies, Carlsbad, CA) based on the manufacturer’s instructions.  Briefly, genomic DNA was mechanically sheared and size fractionated on 0.8% (w/v) agarose gel.  Fragments of 1 to 4 kb were purified from gels, ligated into pCR-Blunt II-TOPO vector, and electroporated into One Shot TOP10 Electrocomp E. coli (Invitrogen).  A total of 7,680 independent clones were selected and grown in individual wells of 96-well round-bottom microtiter plates containing 120 microliter of LB supplemented with 50 microgram/ml of kanamycin.  For amplification of the cloned genomic fragments, 2 microliter of each cell suspension was used as amplification template in 50 microliter PCR reactions containing 200 microM of each dNTP (Takara Mirus Bio Inc., Madison, WI), 2.4 mM MgCl2, 50 pmol of each M13-forward and M13-reverse primers, and 2.5 U of Taq DNA polymerase in 1X PCR buffer (Sigma).  PCR amplifications were performed in 96-well PCR plates, and aliquots of the PCR products were analyzed on agarose gels (with an average size of 1.5 kb).  The remaining amplified mixtures were transferred into 96-well microtiter plates, precipitated by addition of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ice-cold ethanol, washed, and resuspended in 30 microliter of 3X SSC (450 mM NaCl, 45 mM sodium citrate, pH 7.0).  The purified amplicons were spotted onto silanated glass slides (CEL Associates, Inc., Pearland, TX) in 4 subarrays (2 x 2 format), each subarray consisted of 44 x 44 clones using an OmniGrid robotic arrayer (GeneMachine, San Carlos, CA).