Champion pet 200 topo expression kit

cDNA was prepared from RNA isolated from radial nerve tissue extractions (see Mashanov et al. 2014 (link), 2015 (link)). Using this template, we amplified the START domain gene with the primer pair RNIF and RN1R2. This product was purified using the ExoSap (USB) protocol. The PCR product was cloned into the pET 200 TOPO vector for 15 mins at room temperature following the protocol of Champion pET 200 TOPO Expression kit (Invitrogen). Afterwards, the construct was heat-shocked into OneShot TOP10 chemically competent E. coli cells (Invitrogen) and incubated in SOC medium (Invitrogen) at 37° C for 1 hour before plating. We screened for positive insertion using selective plating with ampicillin and Colony PCR, using specific primers supplied by the Champion pET 200 TOPO Expression kit (Invitrogen). All constructs were analyzed in 1% agarose gels run at 120 V for 40 minutes in 10 mM Sodium Borate buffer, visualized with SafeView Classic (Applied Biological Materials) staining in a ChemiDoc XRS+ System. Sanger Sequencing verified bacterial colonies that were positive for STARD10 insertion.