URL: http://www.addgene.org/98750
Proper Citation: RRID:Addgene_98750
Bacterial Resistance: Ampicillin
Defining Citation: PMID:24315440
Vector Backbone Description: Backbone Marker:Zhang lab (Addgene plasmid #42230); Vector Backbone:pX330; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Comments: Because there exists a BbsI cleavage site on CMV-mcherry-pA cassette as well, it is suggested that the BbsI digested product need to be recovered directly instead of agarose gel purification. User Instruction: Digest the px330-mcherry plasmid with BbsⅠ enzyme. After that, run a small amount of the digest on an agarose gel electrophoresis to check the digestion efficiency. If the px330-mcherry plasmid has been well digested, recover the remaining digestion products using a DNA purification kit (such as Tiangen, DP214-03) for restriction enzyme cleanup. In our opinion, any commonly used DNA or PCR purification kit procedure would work well, but not agarose gel purification. We do not suggest combined digestion and ligation. For all other steps (including ligation of annealed oligos into this plasmid), refer to the Zhang lab protocol for pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230).
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Source: Addgene