Mmessage t7 polymerase kit

Manufactured by Thermo Fisher Scientific

The MMessage T7 polymerase kit is a laboratory tool used for in vitro transcription of RNA from DNA templates. The kit contains the T7 RNA polymerase enzyme, necessary buffers, and other reagents required for the RNA synthesis process.

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T7 polymerase mMessage mMachine kit T7 RNA polymerase mMESSAGE mMACHINE T7 kit MMessage T7 kit T7 polymerase T7 kits

6 protocols using mmessage t7 polymerase kit

KCNQ1 Channel Mutagenesis Protocol

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Point mutations of the KCNQ1 channel were engineered using overlap extension and high-fidelity PCR. All primer sequences used in this study can be found in Supplementary Table 1. Each mutation was verified by DNA sequencing. The cRNA of mutants was synthesized using the mMessage T7 polymerase kit (Applied Biosystems-Thermo Fisher Scientific).

Hou P., Eldstrom J., Shi J., Zhong L., McFarland K., Gao Y., Fedida D, & Cui J. (2017). Inactivation of KCNQ1 potassium channels reveals dynamic coupling between voltage sensing and pore opening. Nature Communications, 8, 1730.

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Engineered Point Mutations Verified

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Point mutations were engineered using overlap extension and high-fidelity PCR. Each
mutation was verified by DNA sequencing. cRNA was synthesized using the mMessage T7
polymerase kit (Applied Biosystems).

Zaydman M.A., Kasimova M.A., McFarland K., Beller Z., Hou P., Kinser H.E., Liang H., Zhang G., Shi J., Tarek M, & Cui J. (2014). Domain–domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel. eLife, 3, e03606.

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KCNQ1 and CaM Mutant Generation

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Point mutations were made in KCNQ1 channel and CaM using overlap extension and high-fidelity polymerase chain reaction. DNA sequencing confirmed the presence of all mutants made. Mutant complementary RNA (cRNA) was made with the mMessage T7 polymerase kit (Applied Biosystems–Thermo Fisher Scientific).

Kang P.W., Westerlund A.M., Shi J., White K.M., Dou A.K., Cui A.H., Silva J.R., Delemotte L, & Cui J. (2020). Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening. Science Advances, 6(50), eabd6798.

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Xenopus Oocyte Expression of Ion Channels

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We synthesized the channel cRNA using the mMessage T7 polymerase kit (Applied Biosystems). The cRNA (TREK-1 (hK2P2.1, KCNK2): 2 ng; TREK-2 (hK2P10.1, KCNK10): 0.5 ng; TRAAK (hK2P4.1, KCNK4): 40 ng; Na_V1.5 (SCN5A): 40 ng, equal ratio of α:β₁ subunits; KCNQ1 (KvLQT1): 8 ng; KCNA (Drosophila Shaker): 8 ng) was microinjected (Drummond Nanoject, Broomall, PA) into stage V or VI defolliculated Xenopus oocytes. Injected cells were incubated in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.6) at 18 °C for 2–5 days before performing the experiments.

Kubanek J., Shi J., Marsh J., Chen D., Deng C, & Cui J. (2016). Ultrasound modulates ion channel currents. Scientific Reports, 6, 24170.

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Engineered KV7.1 Channel Mutations

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Point mutations of the K_V7.1 channel were engineered using overlap extension and high-fidelity PCR. Each mutation was verified by DNA sequencing. The cRNA of mutants was synthesized using the mMessage T7 polymerase kit (Applied Biosystems-Thermo Fisher Scientific). All primer sequences used in this study can be found in Supplementary table 1.

Hou P., Kang P.W., Kongmeneck A.D., Yang N.D., Liu Y., Shi J., Xu X., White K.M., Zaydman M.A., Kasimova M.A., Seebohm G., Zhong L., Zou X., Tarek M, & Cui J. (2020). Two-stage electro–mechanical coupling of a KV channel in voltage-dependent activation. Nature Communications, 11, 676.

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KCNQ1 Point Mutation Generation

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Overlap extension and high-fidelity PCR were used for making KCNQ1 channel point mutations. Each KCNQ1 mutation was verified by DNA sequencing. Then cRNA of WT KCNQ1 and mutants were synthesized using the mMessage T7 polymerase kit (Applied Biosystems-Thermo Fisher Scientific) for oocyte injections.

Hou P., Shi J., White K.M., Gao Y, & Cui J. (2019). ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling. eLife, 8, e48576.

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