Dig easy hybtm

Genomic DNA of 10 µg from the cowpea lines of the cassettes AtRps5a pro Cre and AtUbq3 pro lox was digested overnight by SpeI-HF ® and MluI-HF ® (New England Biolabs), respectively. Restriction fragments were separated by gel electrophoresis and then transferred to GeneScreen Plus ® hybridization transfer membrane (Cat# NEF1017001PK, PerkinElmer, Waltham, MA, USA). Hybridization was conducted using a digoxigenin (DIG)-labeled probe targeting either the Cre gene for the cassette AtRps5a pro Cre or the ZsGreen gene for the cassette AtUbq3 pro lox and using DIG Easy Hyb TM as hybridization buffer following the Roche DIG application manual for lter hybridization (Eisel et al. 2008 ). The probes were generated and labeled by PCR with primers p3769/p3770 and p3700/p3701 (Table S3) for Cre and ZsGreen, respectively using the Roche PCR DIG labeling mix following the manufacturer's protocol. Detection was conducted using the chromogenic assay with NBT/BCIP according to the Roche DIG application manual for lter hybridization (Eisel et al. 2008) . All chemicals used for making buffers, as well as blocking reagent (Cat# 11096176001), anti-DIG-AP (Cat# 11093274910), and NBT/BCIP (Cat# 11681451001), were from Millipore Sigma.