Genetailor site directed mutagenesis kit

To create novel alleles in the PrP OR equivalent to the synthetic peptides (sequences available in Supplementary Materials and Methods) examined by electron paramagnetic resonance (EPR) spectroscopy (Supplementary Fig S1), the sequences encoding the N-terminal portion (with the mutations included) of PrP and part of the 5' UTR were synthesized by GenScript Inc. The cassettes encoding the OR-encoding sequences of interest were excised from the plasmid provided by GenScript using KpnI (New England Biolabs), annealed into the full-length PrP sequences in an existing pCDNA3 plasmid using T4 DNA ligase (New England Biolabs), and correct full-length PrP sequences were then moved into another vector pBudgfp by digestion with XbaI and HindIII (New England Biolabs), followed by gel extraction and ligation. For phenylalanine substitutions into the hydrophobic domain, proline substitutions into the octarepeats (G62P, G70P, G78P, G86P) and H95A mutations, primers were designed as per the GeneTailor Site-Directed Mutagenesis kit (Invitrogen) and the mutagenesis reaction was carried out as per the manufacturer's protocol. One microlitre of each reaction was transformed into competent DH5α cells (Invitrogen), and plasmids were isolated using a miniprep kit (Qiagen). Correct plasmids were confirmed by sequencing.

The C-Nap1 CTD (residues 1964–2442) was subcloned into pLeics 20 or pGEX for expression in mammalian cells with an N-terminal Myc tag or bacteria with an N-terminal GST tag, respectively. The rootletin CTD (residues 1651–2018) was fused to an N-terminal maltose-binding protein (MBP) by subcloning into the pMALc2X vector, expressed in bacteria and purified by amylose resin affinity chromatography. Cep135 constructs have been described previously (Kim et al., 2008 (link)). Site-directed mutagenesis was performed using the Genetailor™ Site-Directed Mutagenesis Kit (Invitrogen) according to the manufacturer's instructions. All constructs were verified by DNA sequencing, and bacterial expression and purification were performed according to standard procedures.

Rat Cx32 cDNA subcloned into pIRES2EGFP. To generate Myc/His-tagged constructs, rat Cx32 cDNA was cloned into BamHI/ApaI sites of pcDNA3.1/Myc-His A. The K➔R and K➔Q mutations were generated using Genetailor site-directed mutagenesis kit, as per manufacturer’s recommendations (Invitrogen/Life Technologies) and subcloned into relevant vectors. pIRES2EGFP-Cx32 was used for imaging, BrdU incorporation, and electrophysiology experiments. pcDNA3.1-Cx32-Myc/His was used in all other experiments to facilitate isolation of Cx32 using the Myc and His epitope tags.