QuikChange Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Upstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.
Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Expression Vectors
Plasmid vectors for expressing human OPTN and its mutants (M98K and M98K-D474N) with HA and GFP-tags have been described [, ]. HA-S177A-OPTN and HA-M98K-S177A-OPTN mutants were made by site-directed mutagenesis from HA-OPTN and HA-M98K-OPTN respectively, following the protocol described in QuikChange Site-Directed Mutagenesis Kit (Stratagene/Agilent). S177A-OPTN and M98K-S177A-OPTN were cloned in pEGFP-C3 (Clontech, 6082–1) to produce GFP-tagged OPTN mutants. cDNA of mouse was amplified by RT-PCR using RNA from RGC-5 cells and cloned in pcDNA3.1 and EGFP vectors. GFP-K38A-Tbk1 and HA-K38A-Tbk1 were made by site-directed mutagenesis from GFP-Tbk1 and HA-Tbk1 respectively. The plasmids, pDest-GFP-LC3B and mCherry-GFP-LC3B were kindly provided by Dr. Terje Johansen (University of Tromsø, Tromsø, Norway) []. Adenoviral vectors expressing wild-type OPTN and its M98K, M98K-D474N and E50K mutants have been described earlier [, ].

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Manufacturer protocol

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