Amplifying the target genomic region by PCR
By onThis protocol describes a procedure for amplifying targeted genomic regions using a PCR reaction
AAVS1 Knock-in
By onThis protocol describes the standard procedure to knock-in constructs to the AAVS1 safe harbor locus in hPSCs.
Molecular cloning of SHIP164 plasmids for expression in mammalian cells
By onThis protocol is to help with the molecular cloning of SHIP164 and the sequences of other proteins.
VPS13D DNA plasmid generation
By onThis protocol describes the basic mole cular cloning technique utilized for the generation of VPS13D constructs in https://doi.org/10.1083/jcb.202010004. This protocol and the enzymes included in it are commercialized by Takara Bio.Due to low expression of big DNA constructs (u003e10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: https://doi.org/10.1083/jcb.202010004" . For most of our cloning procedures, Infusion cloning (Takara) was used.
ATG3 construct cloning
By onProtocol describing the cloning of ATG3 construct for production of recombinant (GFP tagged) ATG3 variants.
Generation of CRISPR constructs
By onThis protocol details the procedure of generation of CRISPR constructs.
Constructs and generation of stable cell lines
By onProtocol used to generate stable Flp-In T-REx-HEK 293 cell lines expressing WT or mutant GCase (E326K or L444P) as a V5-FLAG-tagged protein using a tetracycline-inducible system.
Plasmid Construction and Gibson Cloning
By onThis protocol describes plasmid construction as described in Adriaenssens & Nguyen et al., 2023 (BioRxiv)
Plasmid Construction Protocol
By onThis protocol details plasmid construction in general terms for the insertion of genes into the pCAG mammalian expression vector.
Cloning, Protein Expression, and Purification of 20S CPs and Assembly Intermediates
By onThis protocol details methods for cloning, expression, and purification of 20S CPs and assembly intermediates for biochemical and structural analysis.
Genetic Modification of Human Induced Pluripotent Stem Cells (hiPSCs) by Lentiviral Transduction Protocol
By onThis is the protocol for the genetic modification of human induced pluripotent stem cells (hiPSCs) using lentiviral transduction method.
Plasmid construction – A RAB7A Phosphoswitch Coordinates Rubicon Homology Protein Regulation of Parkin- Dependent Mitophagy
By onPlasmid construction via restriction enzyme digest and ligation.
