Spikelets and flag leaves were harvested in 2.0 mL safe lock tube (Eppendorf Co., Ltd., Tokyo, Japan) with zirconia beads and stored at −80 °C until free amino acid extraction.
The remaining Qiazol up to 1 ml was added to the homogenized mixture and the total homogenate was placed in a phase lock tube to separate out the aqueous phase through centrifugation.
Briefly, 500 μl of pre-heated (56°C for 10 minutes) denaturing solution (4 M guanidine isothiocyanate, 0.02 M sodium citrate, 0.5% sarkosyl, 0.1 M β-mercaptoethanol) was added to a 1.5 mL Safe Lock tube extraction tube (Eppendorf, Westbury, NY) containing the stain or swab.
Final sequence-verified plasmids were prepared using a QIAgen HiSpeed Midiprep kit using RNase free reagents and stored in RNase-free Eppendorf Safe Lock Tubes.
PMCA was performed in 0.5 ml Eppendorf safe lock tubes that had been subjected to steam sterilization at 121°C and 3 bar for 20 min prior to use and were sealed with parafilm and paraffin wax before PMCA processing.
RNA was isolated from Trizol by a chloroform extraction, assisted by phase lock tubes.
RNA was extracted with Total RNA Isolation reagent (Sigma) as per the manufacturer's guidelines using phase lock tubes (Eppendorf, Cambridge, UK).
RNA was extracted with TRI reagent (Thermo Scientific, Cramlington, UK) following the manufacturer's guidelines using phase lock tubes (Eppendorf, Cambridge, UK).
RNA extraction used TRIzol® reagent (#15596018; Invitrogen), phase lock tubes (#2302810, 5Prime Inc., Gaithersburg, MD), chloroform C-243232, Sigmand and the PureLink RNA mini-kit (#12182; Invitrogen) with on-column DNA digestion using PureLink™ DNase (#12185010; Invitrogen).
Fourth, samples were treated with 55μL of 5 M NaCl (Sigma, St . Louis MO) and 50μL of prewarmed (to 60°C) 10% Hexadecyltrimethyl-ammonium bromide (Sigma, St . Louis MO) and then incubated at 60°C for 20 min. Fifth, samples were mixed with 470μL of Phenol Chloroform:Isoamyl Alcohol (25:24:1; Sigma, St . Louis MO) and transferred to prespun Phase Lock Tubes (2 mL, heavy; 5 Prime, Gaithersburg, MD).
One-third of all primary PCR reactions from a single 96-well plate were pooled and purified by phenol/chloroform extraction using Phase Lock tubes (Eppendorf, Cambridge, UK) to remove oligonucleotide primers and DNA polymerase that could interfere with hybridization enrichment.
