Acroprep advance 96 filter plate

DNA was isolated from approximately 100 mg of fresh leaf blade + pseudostem tissue for 577 training set mother plants, using a high-throughput method based on that described by Whitlock et al. (2008 (link)) with modifications including a final binding, washing and eluting DNA from AcroPrep™ Advance 96 Filter Plates (Pall Corporation, Ann Arbor, MI, USA). DNA quality was checked via visualisation on ethidium bromide stained 0.8% (wt/vol) agarose/TBE gels and then quantified using the Quant-iT™ PicoGreen^® dsDNA Assay Kit (Invitrogen, Carlsbad, CA). DNA concentrations were normalised to 20 ng/μl and subsequently used for GBS library preparation. GBS libraries were generated following the methodology of Elshire et al. (2011 (link)), with 100 ng of DNA digested using ApeKI (New England Biolabs, Ipswich, MA) and ligated to a unique barcoded adapter and a common adapter (99 ng). A total of six libraries were developed in 96-plex which included a blank and a common positive control sample. Each library was passed through a Pippin Prep™ DNA size selector (Sage Science, Beverly, MA, USA) to isolate fragments between 193 and 313 bp, which were then sequenced on two lanes of an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) at AgResearch Invermay, New Zealand.

To determine the antibody concentration, cell supernatant was filtered through 0.2 μm filter plates (AcroPrep Advance 96 Filter Plates; Pall Corporation) before assessing the antibody titer using the UltiMate 3000 System (Thermo Fisher Scientific) equipped with a Protein A POROS A 20 μm column (Thermo Fisher Scientific). The buffer for column equilibration and antibody binding consisted of 50 mM sodium hydrogen phosphate, 150 mM sodium chloride, titrated with sodium hydroxide to pH 7.5 and the elution buffer of 5.7% phosphoric acid, 150 mM sodium chloride with pH 2.5 (Carl Roth). Finally, protein concentration was detected by UV absorbance at 280 nm.

One hundred-microliter membrane aliquots containing 5 μg (CHO-hA₁AR) or 22 μg (HEK293-hA_2AAR) of protein in assay buffer were harvested by rapid vacuum filtration through 1-μm glass fiber AcroPrep Advance 96 filter plates (Pall Corporation, Ann Arbor, MI, USA) using an extraction plate manifold (Waters, Milford, MA, USA) and a 12-channel electronic pipette (Gilson, Middleton, WI, USA). Filters were subsequently washed three times with ice-cold assay buffer and dried for 1 h at 55 °C. It was essential that the filter plates were completely dry before continuing with ligand elution as described below in “Sample elution.”