lenti sgRNA(MS2)_zeo backbone

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmid construction
Individual gRNAs or minigenes, including ribozymes- or tRNA-linked Dmd, MYBPC3, FST and MYBPC3-DMD 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 (30). The FST 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.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for lenti sgRNA(MS2)_zeo backbone below.

Download manufacturer protocol

Videos

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