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

Plasmids.
BART promoter reporter plasmids were constructed by cloning the inserts from the previously described BART P1 and P2 promoter plasmids () into the pGL2 (Amersham Pharmacia) luciferase reporter plasmid. The LMP1 expression vector was constructed by cloning the coding region of LMP1 into a V5-tagged pSG5 vector. The pcDNA3.1-EGFP (where EGFP is enhanced green fluorescent protein) expression vectors containing NF-κB p50 and p65 and the IκBα expression plasmid containing the IκBα-AA mutation and NF-κB-responsive reporter were kindly provided by Zhenguo Wu (Hong Kong University of Science and Technology, Hong Kong, People's Republic of China) (). NF-κB p65 was also subcloned into a 5′ Flag-tagged pcDNA3.1 vector in this study. For the construction of cell lines stably expressing LMP1, we cloned the LMP1 gene including its 3′ untranslated region (3′ UTR) (GenBank accession number ; nucleotides 167120 to 169474) into the pBABE-Puro vector (Cell Biolabs, Inc.). Full-length and truncated forms of the 3′ UTR of LMP1 (GenBank accession number ; nucleotides 167120 to 168159) were cloned into the cloning site downstream of firefly luciferase in a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). Mutagenesis of the BART promoter and LMP1 was performed using a QuikChange site-directed mutagenesis kit (Stratagene), as described by the manufacturer. Primers are listed in .



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Manufacturer protocol

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

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