pX333-sgNF1-sgTrp53-Cas9

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

Plasmids and adenoviral vectors
The Ad-sgTrp53-Cre adenoviral vector was constructed as follows. The pX333 vector was kindly provided by A. Ventura (Memorial Sloan Kettering Cancer Center). The Cas9 gene in pX333 was replaced by Cre recombinase (Addgene 60229) using AgeI and EcoRI sites by standard cloning methods. For cloning pX333-sgTrp53-Cre, the pX333-Cre vector was digested with BsaI enzyme and ligated to annealed sgRNA oligonucleotides (Supplementary Table 5) targeting mouse Trp53 exon 7. In the final step, pX333-sgTrp53-Cre vector was cloned into the Ad5 adenoviral shuttle vector, which was kindly provided by A. Ventura (Memorial Sloan Kettering Cancer Center) using XhoI and EcoRI sites. The pcDNA3-EGFP vector was purchased from Addgene (13031). Restriction enzymes and T4 ligase were purchased from New England Biolabs. The pX333-sgNF1-sgTrp53-Cas9 adenoviral vector was constructed using a similar approach. Recombinant adenoviruses were generated by Viraquest Inc.



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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pX333-sgNF1-sgTrp53-Cas9 below.

Download manufacturer protocol

Videos

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