pLKO.1 puro

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

Plasmid construction
Individual gRNAs or minigenes, including ribozymes- or tRNA-linked and gRNAs, were synthesized by Integrated DNA technologies (IDT) and inserted into pLKO-GFP-2A-SpCas9, pLKO-mCherry-2A-SpCas9 or pLKO puro-2A-SpCas9 vectors containing two BsmBI restriction sites as previously described (). The gRNAs were cloned into the Lenti_sgRNA(MS2)_zeo plasmid (Addgene, #61427) with modified cloning site. In order to assemble the HH-i20-tRNA-i23-HDV constructs driven by CMV promoter, two minigenes were used as template to produce fusion PCR products and then inserted into pAAV-MCS. The HH-i20-tRNA-i23-HDV cassette was also subcloned into pLKO-gRNA under the U6 promoter or pRNAT-H1.1 with MHCK7 promoter. All gRNA sequences and primers are listed in . All minigenes are listed in .

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pLKO.1 puro below.

Download manufacturer protocol

Videos

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