The coding sequence of rat Atf3 was cloned onto the pcDNA6/myc-His vector (V22120, Thermo Fisher Scientific) for overexpression of Atf3 in mammalian cells. The DNA fragment corresponding to the 3′ UTR of Atf3 mRNA containing the putative binding site for miR-27a-3p was cloned into the psiCHECK2 luciferase vector (C801A, Promega, Madison, WI, USA) for the luciferase assay (CosmoGeneTech, Seoul, Korea). To check whether miR-27a-3p affects Runx2 and Alp mRNA stability, both the 3′ UTR of Runx2 mRNA and the 3′ UTR of Alp mRNA were cloned into the psiCHECK2 luciferase vector.
Pcdna6 myc his vector
Manufactured by Thermo Fisher Scientific
The pcDNA6/myc-His vector is a plasmid vector designed for the expression of recombinant proteins in mammalian cells. It contains a CMV promoter for high-level expression, a C-terminal myc and 6xHis tag for detection and purification, and a blasticidin resistance gene for selection of stable cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Tks gflex dna polymerase, by Takara Bio (2 mentions)
Psicheck 2 luciferase vector, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
P3 flag cmv 10 vector, by Merck Group (1 mentions)
Sanger sequencing, by Takara Bio (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
4 protocols using pcdna6 myc his vector
1
Overexpression and Luciferase Assay for Atf3, Runx2, and Alp
2
Cloning and Validation of HDAC5 Expression
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The coding sequence of rat HDAC5 was cloned onto pcDNA6/myc‐His vector (V22120, ThermoFisher Scientific) for overexpression of HDAC5 in mammalian cells. A DNA fragment corresponding to the 3′UTR of rat HDAC5 containing the putative binding site for miR‐134‐5p was cloned into psiCHECK™‐2 vector (C8021A, Promega) for the luciferase assay.
3
Cloning and Characterization of UBE2D1 and SGK1
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The cDNAs encoding the UBE2D1 and SGK1 proteins were derived from the Genome Network Project (RIKEN BRC) clones HGY088581 and HGX001612, respectively [12] (link). The UBE2D1 cDNA was amplified by Tks Gflex™ DNA Polymerase (TaKaRa Bio, R060A) using the following primer pair: 5'-AAA GCG GCC GCG ATG GCG CT-3' and 5'-CCC GGG ATC CTT ACA TTG C-3'. The resulting PCR product was digested with BamHI and NotI and subcloned into the similarly digested p3×FLAG-CMV10 vector (Sigma-Aldrich, E7658). The SGK1 cDNA was amplified by Tks Gflex™ DNA Polymerase (TaKaRa Bio, R060A) using the following primer pair: 5'-CCC CGG ATC CCC ACC ATG ACG GTG AAA ACT GAG-3' and 5'-GGG CTC GAG GAG GAA AGA GTC CGT GGG-3'. The resulting PCR product was digested with BamHI and XhoI and subcloned into the similarly digested pcDNA™6 myc-His vector (Thermo Fisher Scientific, V22120). The inserted sequences were confirmed by the Sanger sequencing.
4
Cloning and Mutagenesis of PI3K p85
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The expression vector for FLAG-tagged WT EGFR (pIDT-SMART [C-TSC] EGFR-FLAG) was prepared as described previously (81 (link)). The complementary DNA encoding the PI3K p85 protein was derived from the Genome Network Project (RIKEN BRC) clone and amplified by Tks Gflex DNA Polymerase (TaKaRa Bio, R060A) using the following primer pair: 5′-CCC CGG ATC CCC ACC ATG TAC AAT ACT GTT TGG AAT-3′ and 5′-GGG GAG CGG CCG CCA TCG CCT CTG CTG TGC ATA-3’. The resulting PCR product was digested with Bam HI-HF (New England Biolabs, R3136S) and NotI (New England Biolabs, R0189S), and subcloned into the similarly digested pcDNA6 myc-His vector (Thermo Fisher Scientific, V22120).
PCR-based site-directed mutagenesis was performed using PrimeSTAR Max DNA polymerase (Takara Bio, R046A) using the following primer pairs: C146S PI3K p85 forward, 5′-CTG GAA TCT TCA ACT CTA TAC AGA ACA-3′, C146S PI3K p85 reverse, 5′-CTT TTC TTT CCA GAC CTT AGA AGT TGA-3’; C656S PI3K p85 forward, 5′- CAG GGC TCC TAT GCC TGC TCT GTA GTG-3′, and C656S PI3K p85 reverse, 5′- GGC ATA GGA GCC CTG TTT ACT GCT CTC-3’.
The inserted and mutated sequences were confirmed by Sanger sequencing (GENEWIZ).
PCR-based site-directed mutagenesis was performed using PrimeSTAR Max DNA polymerase (Takara Bio, R046A) using the following primer pairs: C146S PI3K p85 forward, 5′-CTG GAA TCT TCA ACT CTA TAC AGA ACA-3′, C146S PI3K p85 reverse, 5′-CTT TTC TTT CCA GAC CTT AGA AGT TGA-3’; C656S PI3K p85 forward, 5′- CAG GGC TCC TAT GCC TGC TCT GTA GTG-3′, and C656S PI3K p85 reverse, 5′- GGC ATA GGA GCC CTG TTT ACT GCT CTC-3’.
The inserted and mutated sequences were confirmed by Sanger sequencing (GENEWIZ).
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