gRNA_Cloning Vector

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

CRISPR/Cas9-mediated gene knockout
The method used for CRISPR/Cas9-mediated gene knockout was described in35,36. In brief, Cas9-target sites for the indicated genes were designed in http://www.genome-engineering.org/crispr/?page_id=41. Then, synthetic nucleotides containing target sites were sub-cloned into gRNA_Cloning Vector (Addgene). Then gRNA vector was co-transfected with hCas9 (Addgene) into L929 cells. After G418 selection, cells were isolated as single clones. The protein quantity of each single clone was analyzed. The Cas9-target sites are: Gβ2-target site-1: TCATCTGAATTCGCCCCAC; Gβ2-target site-2: CTGTGCCTACGCCCCCTCA; Src-target site: GCCGCGGGCGGCACGAAGG.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for gRNA_Cloning Vector below.

Download manufacturer protocol

Videos

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