Pt7 cas9

Manufactured by OriGene

The PT7-Cas9 is a plasmid construct that expresses the Cas9 protein under the control of a T7 promoter. Cas9 is a bacterial-derived RNA-guided DNA endonuclease used in CRISPR gene editing applications.

Automatically generated - may contain errors

Lab products found in correlation

Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (2 mentions) Pcs2 cas9 msa, by Addgene (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Silencer sirna construction kit, by Thermo Fisher Scientific (1 mentions)

2 protocols using pt7 cas9

Cas9 RNA In Vitro Transcription

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Cas9 or Cas9-streptavidin plasmid (this protocol is intended for plasmids containing a T7 promoter)○

Cas9: pT7-Cas9 (available commercially from OriGene Cat. No. GE100014)

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Cas9-streptavidin: PCS2+Cas9-mSA (available from Addgene Plasmid No. 103882)○

NOTE: The SP6 promoter in this plasmid was replaced with a T7 promoter using standard cloning techniques.

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NOTE: Protocol can be adapted to use a plasmid containing an SP6 promoter.

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Restriction enzyme appropriate to linearize Cas9 plasmid (see note 1c)

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Phenol/Chloroform

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3 M NaOAc, pH 5.3

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100% ethanol

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mMessage mMachine T7 Ultra kit (Thermo Fisher Cat. No. AM1345)•

NOTE: Use SP6 in vitro transcription kit if using a plasmid containing an SP6 promoter.

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RNeasy Mini Kit (Qiagen Cat. No. 74104)

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Dry ice

Pineault K.M., Novoa A., Lozovska A., Wellik D.M, & Mallo M. (2019). Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse. MethodsX, 6, 2088-2100.

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Microinjection of KDM6A and KDM6B mRNA/siRNA in Mouse Oocytes

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The coding region of KDM6A and KDM6B was amplified from mouse tail‐tip genome. Forward and reverse primers contained T7 promoter and HA sequences, respectively. To prepare mRNAs for microinjection, pT7‐Cas9 (OriGene, GE100014), KKDM6A and KDM6B expression vectors were linearized and subjected to phenol–chloroform extraction and ethanol precipitation. The mRNA synthesized with the mMESSAGE‐mMACHINE T7 Ultra Kit (Thermo, USA) according to the manufacturer's instructions. Two different siRNA species targeting KDM6B were designed and synthesized using the Silencer siRNA Construction Kit (Ambion, USA) following the manufacturer's instructions. A commercially available siRNA without any specificity to known genes was used as control. As previously described 41, with minor modifications, 8 pl of siRNA or mRNA was microinjected into the cytoplasm of denuded oocytes. Oocytes were injected using Piezo‐operated blunt‐end micropipette (3–5 μm internal diameter). After injection, oocytes were kept at RT for 30 min and then moved into the incubator.

Yang L., Song L., Liu X., Bai L, & Li G. (2018). KDM6A and KDM6B play contrasting roles in nuclear transfer embryos revealed by MERVL reporter system. EMBO Reports, 19(12), e46240.

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