pSpCas9(BB)-2A-Puro (PX459) V2.0

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

2.2. Plasmids
HA-tagged mouse β3AR coding sequence was amplified by PCR from pGL2-HA-mβ3AR construct, a kind gift from Dr. Sheila Collins [25] and cloned into pcDNA3.1 vector. Ser247-mutated β3AR construct was generated by QuikChange site-directed mutagenesis kit from Agilent Technologies (#200519) based on pcDNA3.1-HA-mβ3AR. CRISPR/Cas9 vector (PX459) was ordered from Addgene (Plasmid #48139). β3AR-targeted Crispr sgRNAs (sense: 5′-CACCGGGCAGAGTCCACCGCTCAAC-3’; antisense: 5′-AAACGTTGAGCGGTGGACTCTGCCC-3′) were synthesized by Integrated DNA Technologies and cloned into PX459 following the protocol provided by Dr. Feng Zhang [26]. Constitutively active ERK2 (ERKca) expression construct was previously described [27].

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) V2.0 below.

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Videos

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