px330-mcherry

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

pX330-mCherry-gHOTAIR Plasmid Construction
The sgRNA design was based on the CRISPR design (http://crispr.mit.edu/) or CHOPCHOP (https://chopchop.rc.fas.harvard.edu/). The pX330-mCherry plasmid expressing hSpCas9 and sgRNA was modified based on the pX330: hSpCas9 + chimeric guide RNA vector (Addgene plasmid # 42230). The sgRNA oligos targeting the upstream and downstream regions of the HOTAIR gene were synthesized and cloned into the BbsI site of pX330-mCherry (pX330-mCherry-HOTAIR) as described (43). Then, the plasmids were transformed into Stbl3 competent cells and sequenced using the U6 sequencing primer.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for px330-mcherry below.

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Videos

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