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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Generation of ERαY537S and ERαD538G cell lines
Our protocol was loosely based on previous work46. Two guide sequences (Supplementary Fig. S1) were cloned into pSpCas9(BB)-2A-Puro(PX459) (Addgene, MA). An HDR template with mutations was cloned into pUC18, and linearized before transfection. Two plasmids with guide sequences and the HDR template carrying either the ERαY537S or ERαD538G mutation were co-transfected into T47D cells. After 3-day selection in 2.5 μg/ml puromycin, followed by 3 weeks of E2-free growth, colonies were transferred to a 24-well plate. Clones were genotyped at the DNA and mRNA levels using PCR and restriction enzyme digestion. Sequencing confirmed selected clones.

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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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