Megaprime kit

Rhizobium plasmids were visualized by the Eckhardt procedure [12 (link)]. Gels were transferred onto Hybond N⁺ membranes (Amersham) using the manufacturer’s protocol and cross-linked using a UV cross linker unit (Stratagene). Hybridizations were performed overnight using α³²P-dCTP-labelled probes (Megaprime kit; Amersham) under high-stringency conditions (65 °C in rapid-Hyb buffer, Amersham). Hybridization signals were detected with a PhosphorImager (Molecular Dynamics).

Southern blotting was performed using standard methods. A 538 bp probe binding the region over MCS-R2 (chr16: 163380—163907 on build hg19) was generated by PCR, using primers FWD 5′-AACAAGAAAACCAGCAGGCTCC-3′ and REV 5′-TGTAAGTCCATCCAGGTGTGAGTTC-3′. This was cloned into the vector pGEM-T-easy (Promega) for confirmation by sequencing prior to excision and purification. About 50 ng of probe was end-labeled with α-P³² using the megaprime kit (Amersham, RPN1604) and hybridized to blotted genomic DNA from patient MC, RC, and a normal control, digested using BamH1. A labeled fragment size of 19 kb is expected when hybridized with the MCS-R2 probe.