Xl10 gold ultracompetent escherichia coli cells

All the DNA constructs used in this study were expanded using the XL10-Gold ultracompetent Escherichia coli cells (Agilent Technologies) and were purified using HiSpeed Plasmid Midi Kit (Qiagen) according to manufacturer’s recommendations.

The 1,410-nucleotide open reading frame encoding the C-terminal 347 amino acid of isoform 1 (NP_008910.2) of ZIP7/SLS39A7 was cloned into vector pXP1510 (lentiviral vector with G418 selection marker and EF1α promoter driving open reading frame expression). Schematic of pXP1510 cloning vector is shown in Supplementary Fig. 16.
Error-prone PCR-mediated mutagenesis was performed using the Ex Taq hot start enzyme (TaKaRa Hot Start Ex Taq Kit, Clontech) in the presence of 100 μM manganese chloride to induce mutations in the ZIP7 open reading frame. Briefly, 100 ng of plasmid DNA (pXP1510-ZIP7) was mutagenized by PCR according to the manufacturer’s protocol using the following primers: XPO2011-F, 5′ -CAGATCCAAGCTGTGACC-3′, and XPO2010-R, 5′ -GTAACGTCTACGTGTCTG −3′. PCR products were separated by gel electrophoresis and purified using a Qiaquick PCR purification kit (Qiagen). After restriction enzyme digest (NotI-HF, AscI, DpnI), the DNA was purified with the PCR purification kit (Qiagen) mentioned above and cloned into pXP1510. Mutagenized DNA was transformed into Escherichia coli XL10-Gold ultracompetent cells (Agilent). A total of 48 colonies were chosen for plasmid sequencing to infer the mutation rate of the library, which was 0.7 non-silent mutations per kilobase.