pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Plasmid constructs
ERT2CreERT2 Was PCR amplified using pCAG-ERT2CreERT2 (Addgene # 13777) as a template and
was cloned between XhoI and NotI sites of the bicistronic pEFIRES vector (31) harboring a
selectable marker conferring resistance to G418.
To generate pX330-5A1, a 19-nt guide sequence (Table 1) targeting exon 1 of the mouse eIF5A1
gene was ligated into a pX330 hSpCas9 plasmid (Addgene # 42230).
pE-eIF5A1smut, containing a FLAG-tagged eIF5A1 that is refractory to the guide RNA, was
constructed in two steps; first, eIF5A1 cDNA was amplified by PCR using a 5’ primer containing a
LoxP site and appending a FLAG tag to the N-terminus of eIF5A1 and a 3’ primer containing a LoxP
site. The resulting fragment was cloned into the bicistronic pEFIRES vector containing a selectable
marker conferring puromycin resistance (Table 2). Next, three silent mutations were introduced at the
site of recognition by the guide RNA to make it refractory to editing by the CRISPR-Cas9 (Table 2).

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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.

Download manufacturer protocol

Videos

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