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

Site-directed mutagenesis and stable line production
Site-directed mutagenesis of Rab11-FIP2 in the pEGFP vectors was performed using Pfu Turbo polymerase according to the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA) with 16-min extension time. Primers were synthesized (Invitrogen, Carlsbad, CA) with one nucleotide change per oligonucleotide sequence. Previously the TCA encoding for amino acid 227 was change to GCA for the S227A mutant (); for the S227E mutant this GCA was mutated to GAA. Production of the doxycycline-inhibitable GFP-Rab11-FIP2(S227E) MDCK cell line was as described to Rab11-FIP2-WT and -S227A (). Briefly, the GFP-Rab11-FIP2(S227E) was cut out of the pEGFP vector (Clontech, Mountain View, CA) with I and I and ligated into the pTRE2hyg vector (Clontech) cut with I and RV. T23 MDCK cells were transfected with Effectene (Qiagen, Valencia, CA) and plated in normal media. The next day the cells were trypsinized and replated in serial dilution with 0.25 ng/ml hygromycin for selection and 20 ng/ml of doxycycline for suppression of enhanced GFP (EGFP) expression. Multiple clones were selected, expanded, and initially characterized; all exhibited similar patterns and levels of EGFP-Rab11FIP2(S227E) expression. One clone was selected to use as the tetracycline-repressible stable cell line.

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Manufacturer protocol

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