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Bbsi pgkpuro2azsg w

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BbsI-PGKpuro2AZsG-W is a plasmid that includes a puromycin resistance gene and a green fluorescent protein (GFP) reporter gene. The plasmid can be used for selection and monitoring of transduced cells.

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Lab products found in correlation

Polybrene, by Santa Cruz Biotechnology (2 mentions) Pgkpuro2amcherry w, by Addgene (2 mentions) One shot stbl3 chemically competent escherichia coli, by Thermo Fisher Scientific (2 mentions) Pspax2, by Addgene (2 mentions) Pmd2 g, by Addgene (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions)

2 protocols using bbsi pgkpuro2azsg w

1

CRISPR gRNA Plasmid Cloning and Lentivirus Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
gRNA oligonucleotides were phosphorylated, annealed and ligated into BbsI-linearized pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W (Addgene, #67975) or pKLV2-U6gRNA5(BbsI)-PGKpuro2AmCherry-W (Addgene, #67977) vectors. One Shot Stbl3 Chemically Competent Escherichia coli (Invitrogen, C7373-03) were used for transformation and Qiagen kits for the purification of the plasmids. Virus was produced with HEK293T cells transfected with psPAX2 (Addgene, 12260), pMD2.G (Addgene, 12259) and the gRNA plasmid using polyethylenimine (PEI, Polysciences, 23966-1). Adherent target cells were seeded the day before transduction with the virus and the help of 8 μg/ml polybrene (Santa Cruz, SC-134220). Suspension cells were seeded in medium with polybrene, virus was added and the cells were spin transduced for 45 min at 2,000 rpm. Final gRNAs are listed in Supplementary Table 1. Samples were measured with a BD LSRFortessa™ flow cytometer (BD Biosciences) using B/E Alexa Fluor 488 (GFP) and YG/D PE Texas Red (mCherry) 4 d, 10 d and 14 d after transduction.
Schick S., Rendeiro A.F., Runggatscher K., Ringler A., Boidol B., Hinkel M., Májek P., Vulliard L., Penz T., Parapatics K., Schmidl C., Menche J., Boehmelt G., Petronczki M., Mueller A.C., Bock C, & Kubicek S. (2019). Systematic characterization of BAF mutations provides insights into intra-complex synthetic lethalities in human cancers. Nature genetics, 51(9), 1399-1410.
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2

CRISPR gRNA Plasmid Cloning and Lentivirus Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
gRNA oligonucleotides were phosphorylated, annealed and ligated into BbsI-linearized pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W (Addgene, #67975) or pKLV2-U6gRNA5(BbsI)-PGKpuro2AmCherry-W (Addgene, #67977) vectors. One Shot Stbl3 Chemically Competent Escherichia coli (Invitrogen, C7373-03) were used for transformation and Qiagen kits for the purification of the plasmids. Virus was produced with HEK293T cells transfected with psPAX2 (Addgene, 12260), pMD2.G (Addgene, 12259) and the gRNA plasmid using polyethylenimine (PEI, Polysciences, 23966-1). Adherent target cells were seeded the day before transduction with the virus and the help of 8 μg/ml polybrene (Santa Cruz, SC-134220). Suspension cells were seeded in medium with polybrene, virus was added and the cells were spin transduced for 45 min at 2,000 rpm. Final gRNAs are listed in Supplementary Table 1. Samples were measured with a BD LSRFortessa™ flow cytometer (BD Biosciences) using B/E Alexa Fluor 488 (GFP) and YG/D PE Texas Red (mCherry) 4 d, 10 d and 14 d after transduction.
Schick S., Rendeiro A.F., Runggatscher K., Ringler A., Boidol B., Hinkel M., Májek P., Vulliard L., Penz T., Parapatics K., Schmidl C., Menche J., Boehmelt G., Petronczki M., Mueller A.C., Bock C, & Kubicek S. (2019). Systematic characterization of BAF mutations provides insights into intra-complex synthetic lethalities in human cancers. Nature genetics, 51(9), 1399-1410.
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