The H19 sequence was synthesized by GenScript and cloned into the pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies). An expression vector encoding the wild-type sequence of the human DMD ZnF domain was purchased from the shRNA and ORF core of MD Anderson Cancer Center, and the coding regions were subcloned into the Gateway™ pET-DEST40 vector for mammalian expression and Gateway™ pET-DEST42 vector for prokaryotic expression (Invitrogen). All single-point and deletion mutations were generated using QuikChange™ Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). DMD ZnF Y/F-A mutant substitutes tyrosine (Y) or phenylalanine (F) to alanine (A) at sites Y3326, F3332, Y3334, and F3341. Recombinant DMD WT and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific). Plasmid transfections were performed using Lipofectamine3000 (Life Technologies) or electroporation using the 4D-Nucleofector™ System (Lonza) according to the manufacturer’s instructions.
Hispur cobalt resin kit
Manufactured by Thermo Fisher Scientific
The HisPur Cobalt Resin Kit is a purification system designed to capture and purify histidine-tagged proteins. It utilizes a cobalt-based resin to selectively bind and isolate the target proteins, allowing for efficient separation and recovery.
Automatically generated - may contain errors
Lab products found in correlation
Gateway pet dest42 vector, by Thermo Fisher Scientific (3 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (3 mentions)
Pgem 3z vector, by Promega (3 mentions)
Bl21 codonplus de3 ripl, by Agilent Technologies (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (3 mentions)
4d nucleofector system, by Lonza (3 mentions)
Gateway system, by Thermo Fisher Scientific (2 mentions)
Scramble sirna and sirna targeting trim63, by Santa Cruz Biotechnology (2 mentions)
Orius 832 camera, by Ametek (1 mentions)
Jem 1400 plus tem, by Ametek (1 mentions)
4 protocols using hispur cobalt resin kit
1
Synthesis and Characterization of DMD ZnF Mutants
2
Molecular Cloning and Expression of DMD
3
Molecular Cloning and Expression of DMD
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H19 DNA sequences were synthesized by GenScript and cloned into pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies) or pMS2 vector for mammalian expression. The full-length human or mouse DMD and human DMD zinc finger domain sequence was obtained from MDACC shRNA and ORFeome Core and Addgene. Gateway™ pET-DEST42 vector (Invitrogen) was used for prokaryotic expression of human DMD c-termini. Mammalian full-length DMD, zinc finger domain of DMD, or H19 wild type and mutant vectors were constructed by subcloning the corresponding gene sequences into the SFB-tagged expression vector (provided by J. Chen, MD Anderson Cancer Center, USA) using the Gateway system (Life Technologies). All single-point and deletion mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Scramble siRNA and siRNA targeting TRIM63 were obtained from Santa Cruz Biotechnology. Plasmid transfections were performed using Lipofectamine3000 (Life Technologies), or electroporation using the 4D-Nucleofector™ System (Lonza) according to manufacturer’s instructions. Recombinant DMD wild type and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific).
4
Visualizing the HP1γ Dimer Structure
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For visualizing the shape and contour of the HP1γ dimer, we produced and purified an N-terminal 6×His-tagged recombinant form of this protein using the pET vector system (Novagen, CA). The HP1γ-encoding plasmid was grown in DE3 BL21 bacteria cells overnight and induced with 0.5 mM IPTG for 90 min at 32 °C. The recombinant protein was purified using the Thermo Scientific HisPur Cobalt Resin Kit according to the manufacturer’s instructions. Protein was dialyzed overnight and concentrated to a final concentration of 1 mg/ml. For visualization at the electron microscopy level, 10 μl of the purified protein solution was placed on the surface of glow-discharged formvar carbon-coated grids. After 30 s, the grids were blotted and stained for 30 s in 1 % uranyl acetate. Micrographs were acquired using a JEOL, JEM-1400Plus TEM at 80-kV accelerating voltage, equipped with a Gatan Orius 832 camera.
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