Illustra quickprep micro mrna purification kit

The mouse antibody Fv sequences were cloned using 5′RACE with modified primers and conducted as described previously33 34 (link). The primers are listed in Table 2. To prepare cDNA templates, mRNA was extracted from hybridoma cells with Illustra QuickPrep Micro mRNA Purification Kit (GE Healthcare, Buckinghamshire, UK). Five hundred nanograms of mRNA were reverse-transcribed into first strand cDNA with SuperScript III First-Strand Synthesis Supermix (Life Technologies, Grand Island, NY). The reaction mix was then treated with RNase H (NEB, Ipswich, MA) and processed for PCR purification (QIAquick PCR purification kit, Qiagen). Poly-dC was added by terminal transferase (NEB) to the 3′ end of the first strand cDNA (corresponding to the 5′ end of the original mRNA). The products were purified with a QIAquick PCR purification kit again before PCR reactions. The 5′RACE was performed using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific, Cincinnati, OH). The Fv fragments were cloned into the pCR4-TOPO vector with a TOPO TA Cloning Kit (ThermoFisher Scientific) for sequencing according to manufacturer’s instructions. Positive clones were screened with Taq DNA polymerase. BigDye (Invitrogen) was used for sequencing according to manufacturer’s instructions.

The cDNA library was constructed by RNA isolation of 100 mM NaCl-treated hydroponically grown 1-week-old KDML105 rice using TRI Reagent® (MRC, USA) and mRNA purification using the Illustra™ Quick Prep Micro mRNA Purification Kit (GE Healthcare, UK). cDNA synthesis and cDNA library construction were carried out using the Uni-ZAP XR vector system (Stratagene, USA). The cDNA was ligated to XhoI-EcoRI double-digested Uni-ZAP XR vector, and then the ligated mixture was packaged using Gigapack III packaging extract (Stratagene, USA). The packaging was performed using XL1-Blue MRF’ E. coli cells, and the cells were cultured on NZY agar plates overlaid with NZY agar containing IPTG and X-gal for blue-white colony selection. The libraries were propagated in XL1-Blue MRF′ E. coli, and the propagated libraries were stored in 0.3% (v/v) chloroform at 4 °C.

DNA of A. aurita mucus (1 mL v/v) and polyps (10 polyps for each preparation) was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, USA). mRNA of 20 polyps was isolated using the illustra™ QuickPrep Micro mRNA Purification Kit (GE Healthcare, Chalfont St. Giles, UK). Fosmid DNA was isolated from 5 mL overnight cultures using High-Speed-Plasmid-Mini Kit (Avegene, Taipei, Taiwan).