QuikChange II XL 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

Plasmids
Mig-RI-c-Myb-HA and Mig-RI-Δ(358–452)c-Myb were obtained as described.

Cells and culture conditions
293T cells and parental and derivative K562 cell lines were maintained as described. Derivative K562 cell lines (Δ(358–452)c-Myb, p89, c-Myb shRNA or c-Myb shRNA expressing p89_shMUT or p75_shMUT) were established by retroviral infection and isolation of green fluorescent protein-positive cells or selection with puromycin.

Transient transfection and dual-luciferase reporter assay
The Mig-RI Δ(358–452)c-Myb or p89 (3 μg) was co-transfected with cyclin B1-Luc-pGL3 or SLUG-Luc-pGL3 (1 μg) and Renilla luciferase (0.02 μg) into 10 K562 cells using nucleofector kit V (Amaxa; Lonza Group, Basel, Switzerland) according to the manufacturer's protocol. After 48 h, cells were diluted 1:5 in fresh medium and cultured for an additional 6–8 h. Cells were then lysed (100 μl of passive lysis buffer; Promega, Madison, WI, USA) and Firefly and Renilla luciferase activities were measured on a luminometer using the dual-luciferase reporter assay kit (Promega). For the assay in 293T cells, cells were seeded into six-well plates at 5 × 10 cells per well. The following day, DNA (5 μg of reporter plasmid, 5 μg of effector plasmid and 0.5 μg of Renilla luciferase) was transfected using ProFection Mammalian Transfection System-Calcium Phosphate (Promega) following the manufacturer's protocol. Cells were washed 16 h after transfection and incubated in fresh medium for an additional 24 h. At 40 h post transfection, cells were lysed in 500 μl of passive lysis buffer per well (Promega) and Firefly and Renilla luciferase activities were measured as described. All the measurements were performed in triplicate.

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Manufacturer protocol

Download the product protocol from Agilent Technologies for QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn below.

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