pX330-U6-Chimeric_BB-CBh-hSpCas9

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

Expression Vector
The plasmid px330-U6-Chimeric-HsPCas9 (Addgene plasmid #42230) was used for expression of the gRNA and of dCas9. It contains two BbsI restriction sites for insertion of an oligonucleotide, which selects the target sequence of the gRNA. This gRNA is under the control of the U6 promoter. The plasmid pAC154-dual-dCas9-VP160-sgExpression (Addgene plasmid #48240) was used for expression of the gRNA and dCas9-VP160. It contains two BbsI restriction sites for insertion of an oligonucleotide, which selects the target sequence of the gRNA. This gRNA is under the control of the U6 promoter. This plasmid was modified to include a gene encoding resistance to puromycin, the latter is separated from the gene encoding dCas9 via a sequence coding for the 2A peptide. There is a nuclear localization sequence (NLS) fused with the dCas9-VP160 gene.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

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Videos

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