Top10 chemically competent escherichia coli

Contig sequences for the investigated genes were identified in the transcriptome dataset by bidirectional BLAST [24 (link)]. Whole transcripts or fragments were amplified by PCR with specific primers (Fw = AGTTTGGGATGGTGGG, Rv = TTCTGGGCTAGCTGGT) from cDNA prepared with SuperScript III (Invitrogen, Waltham, MA, USA), ligated into pgemT-easy vector (Promega, Madison, WI, USA) and cloned into Top10 chemically competent Escherichia coli (Invitrogen). Clone sequences were verified by Sanger sequencing. DIG-labelled sense and antisense RNA probes were generated from plasmid DNA with T7- and SP6-RNA polymerases (Roche, Madison, WI, USA).

The 3′-untranslated region (UTR) of RAD1 (Medtr4g104020) was identified in the M. truncatula v 4.0 genome release and supported by RNAseq coverage (http://jcvi.org/medicago/). A 177 bp fragment of the 3'UTR was amplified from M. truncatula A17 cDNA using Phusion DNA polymerase (New England Biolab Inc., UK) with primers listed in Supplementary Table S1. This sequence was unique in the Medicago genome and did not return any blast hit other than the query. Amplicons were immediately ligated into the pENTR/D-TOPO Cloning Kit (Invitrogen) used as an entry Gateway vector, and transformations were carried out into TOP10 chemically competent Escherichia coli (Invitrogen). Minipreps were carried out using a QIAprep Spin Miniprep Kit (QIAGEN). Then recombination was performed into a pK7GWIWG2_II-Red Root vector (VIB, Ghent University, Belgium) using Gateway LR Clonase II Enzyme Mix (Invitrogen) to produce hpRAD1 binary constructs. A control recombination with the pENTR-GUS clone provided with the LR kit was performed, and the resulting vector was used as a control in the experiment and designated hpuidA.