Plasmids encoding MIM-GFP and GFP were prepared as described previously [16 (link)]. pIRTKS-GFP was synthesized and cloned into the vector pEZ-M98 by Genecopoeia (Gaithersburg, MD). pIRTKSΔSH3, which encodes a protein with a deletion of the SH3 domain (aa 342-399), was prepared by using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) using pIRTKS-GFP as the template. To prepare pIRTKS-I-BAR-MIM-CT-GFP, a DNA sequence that has an open reading frame corresponding to the human IRTKS protein from aa 1 to 231 and human MIM protein from aa 235 to 755 was synthesized by GeneScript (Nanjing, China) and cloned into pUC57 vector. This DNA sequence is also flanked by a HindIII site at the 5′ and a BamH1 site at the 3′, respectively. After digestion with HindIII and BamH1, the insert was subcloned into pEZ-M98. To prepare pMIM-I-BAR-IRTKS-CT-GFP, a DNA sequence that is flanked by HindIII and BamH1 sites and has an open reading frame corresponding to human MIM protein from aa 1 to 250 and human IRTKS protein from aa 250 to 511 was synthesized by GeneScript. This DNA fragment was also subcloned into pEZ-M98 as above. For all the mutants, the fidelity of the mutations was confirmed by DNA sequencing.
Pez m98
Manufactured by GeneCopoeia
Sourced in China
The PEZ-M98 is a compact and versatile pipetting robot designed for automated liquid handling in life science laboratories. It features precise liquid transfer capabilities and can handle a wide range of sample volumes. The PEZ-M98 is suitable for various applications in genomics, proteomics, and cell biology research.
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Lab products found in correlation
2 protocols using pez m98
1
Cloning and Characterization of IRTKS-MIM Fusion Proteins
2
Knockdown and Overexpression of TMEM40
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The specific small interfering RNA of TMEM40 and a scrambled negative control were purchased from RiboBio (Guangzhou, China). The overexpression vector pEZ-M98 and a scrambled negative control empty vector were also purchased from GeneCopoeia (Guangzhou, China). The shRNA vector and a scrambled negative control vector were obtained from Genechem (Shanghai, China). The sequence of siRNA was 5′-GGAUGAGCUUCAACUCUAUTT-3′; NC was 5′-UUCUCCGAACGUGUCACGUTT-3′; shRNA was 5′-GUGGACGCCUCUCAGUUAA-3′; NC was 5′-TTCTCCGAACGTGTCACGT-3′;
After cells were cultured 24 h, the cells were transiently transfected with corresponding siRNA or plasmid DNA using Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with siRNA or shRNA and plasmid were harvested for qRT-PCR or western blot detection.
After cells were cultured 24 h, the cells were transiently transfected with corresponding siRNA or plasmid DNA using Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with siRNA or shRNA and plasmid were harvested for qRT-PCR or western blot detection.
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