pcDNA-mC/EBPb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmids and Stable Overexpression
3T3-L1 clones, which stably overexpress C/EBPβ, were generated with retrovirus transduction (Platinum Retrovirus Expression System, Pantropic, Cell Biolabs, San Diego, CA). pcDNA-mC/EBPb was a gift from Jed Friedman (Addgene plasmid #49198). The plasmid was digested with and , and subcloned into the retroviral vector, pMXs-IRES-Puro using the - site. The plasmids were transfected to the packaging cell line, and the culture supernatants were collected 24 and 48 hr later, passed through 0.22-μm filters, and added to the cells. Drug selection by 2 μg/ml puromycin was initiated 72 hr after infection. For CRISPR-Cas9 system, pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230). Hif1a target sgRNAs were designed by using CRISPRdirect, and ligated into pX330 plasmid by following the procedures from Zhang lab (http://www.genome-engineering.org/crispr). Sequences are listed in Table . 3T3-L1 cells were co-transfected with the two pX330-Hif1a and a puromycin resistant plasmid which facilitates selection with puromycin (2 μg/ml). Selected colonies went further selection by HREluc activity under hypoxia.

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pcDNA-mC/EBPb below.

Download manufacturer protocol

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