pCAG-GFP (Addgene plasmid 11150) (Matsuda and Cepko, 2004 (link)). pCAG-YFP (Addgene plasmid 11180) (Matsuda and Cepko, 2004 (link)). pCAG-DsRed (Addgene plasmid 11151) (Matsuda and Cepko, 2004 (link)). pRho-GFP-IRES-AP (referred to as Rho-GFP) (Emerson and Cepko, 2011 (link)). pCAG-nlacZ (Cepko lab, Harvard Medical School) pCAGEN (Addgene plasmid 11160) (Matsuda and Cepko, 2004 (link)). pCALNL-DsRed (Addgene plasmid 13769) (Matsuda and Cepko, 2004 (link)). pCAFNF-DsRed (Addgene plasmid 13771). (Matsuda and Cepko, 2004 (link)). pCALNL-luc2 (Tang et al., 2015 (link)). pRL-TK (#E2241; Promega, Madison, WI).
Pcag dsred
Manufactured by Addgene
Sourced in United States
The PCAG-DsRed is a plasmid designed for expression of the DsRed protein in mammalian cells. The plasmid contains the DsRed gene under the control of a cytomegalovirus (CMV) promoter, which drives high-level expression of the protein in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Pcag gfp, by Addgene (4 mentions)
Pcag yfp, by Addgene (4 mentions)
Pcalnl dsred, by Addgene (3 mentions)
Prl tk, by Promega (3 mentions)
Pcag cfp, by Addgene (3 mentions)
Pcagen, by Addgene (1 mentions)
Ej2gfp, by Addgene (1 mentions)
Pcbascei, by Addgene (1 mentions)
Fugene 6, by Promega (1 mentions)
Anti tuj1, by BioLegend (1 mentions)
Anti tbr1, by Abcam (1 mentions)
Anti ctip2, by Abcam (1 mentions)
Anti h3, by Cell Signaling Technology (1 mentions)
Anti dsred, by Takara Bio (1 mentions)
Anti foxp1, by Abcam (1 mentions)
Anti cre, by Merck Group (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
9 protocols using pcag dsred
1
Versatile Fluorescent Protein Expression Plasmids
2
Diverse Fluorescent Protein Expression Vectors
3
MMEJ Efficiency Quantification in PARP Inhibition
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In the reporter assay, 2.5 × 105 cells were transfected with siControl and siRB for 3 days. Cells were then treated with DMSO, 1 μM or 10 μM olaparib for 1 h in both siControl- and siRB-treated cells. Cells were then transfected with the EJ2GFP (Addgene, Watertown, MA, USA) to quantify MMEJ efficiency. Furthermore, 1 μg EJ2GFP plasmids was transfected along with 1 μg pCBASceI (Addgene, Watertown, MA, USA) to cells with Fugene6 (Promega, Madison, WI, USA). The ratio of Fugene6 to the transfected plasmid was 3:1. In another round of transfection, the same numbers of cells transfected with 2 μg red fluorescent protein (RFP) expression plasmid pCAG-DsRed (Addgene, Watertown, MA, USA) were used to normalize the transfection efficiency [27 (link)]. Cells were then incubated for 72 h after transfection and collected for flow cytometry analysis.
4
Molecular Markers for Neuronal Lineages
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Plasmid pCAG-dsRed was from Addgene (Plasmid #11151). Neurod1-Cre was provided by Dr. Franck Polleux (Columbia University). Pak3K297R was gifted by Dr. Rick Horwitz (University of Virginia). The PAK3 mutant was subcloned into the pCMVmyc vector.
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
The following primary antibodies were used: anti-EZH2 1:500 (BD Biosciences, San Jose, USA, Cat. No. 612666), anti-EZH2 1:1000 (Cell Signaling, Danvers, USA, Cat. No. #5246), anti-GAPDH 1:5000 (Sigma-Aldrich, St. Louis, USA, Cat. No. G8795), anti-GFP 1:1200 (Thermo Fisher Scientific, Waltham, USA, Cat. No. A10262), anti-TUJ1 1:1000 (Biolegend, San Diego, USA, Cat. No. 801201), anti-H3K27me3 1:2000 (Millipore, Burlington, USA, Cat. No. 07449), anti-H3 1:1000 (Cell Signaling, Cat. No. #4499), anti-dsRED 1:1000 (Clontech, Mountain View, USA, Cat. No. 632496), anti-Tbr1 1:1000 (Abcam, Waltham, USA, Cat. No. ab31940), anti-Ctip2 1:500 (Abcam, Cat. No. ab18465), anti-Foxp1 1:1000 (Abcam, Cat. No. ab16645), and anti-Cre 1:1000 (Millipore, Cat. No. MAB3120).
5
Fluorescent Plasmid Validation Protocol
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Plasmids encoding fluorescent reporter proteins were used to validate electroporation: pCAG‐DsRed (Addgene: 11151), pCAG‐YFP (Addgene: 11180), pCAG‐EGFP (Addgene: 11150), pCAG‐CFP (Addgene: 11179), pCBA‐TdTomato (Addgene: 28017). Plasmids were amplified and purified according to the suppliers’ recommendations, filtered, and stored at −20°C until use.
6
Diverse Fluorescent Protein Expression Vectors
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pCAG-GFP (Addgene plasmid 11150) 39 (link), pCAG-YFP (Addgene plasmid 11180) 39 (link), pCAG-CFP (Addgene plasmid 11179) 39 (link), pCAG-tdT (Cepko lab, Harvard Medical School), pCAG-mCherry (Cepko lab, Harvard Medical School), pCAG-DsRed (Addgene plasmid 11151) 39 (link), pRL-TK (Promega, #E2241), pRho-GFP-IRES-AP (referred to as Rho-GFP in main text) 40 (link), pCAG-nlacZ (Cepko lab, Harvard Medical School). pAAV-CAG-FLEX-tdT was a gift from Edward Boyden (Addgene plasmid #28306), pCALNL-DsRed (Addgene plasmid #13769) 41 (link).
7
In Utero Electroporation of Plasmids
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Plasmids used in this study were as follows: pCag-DsRed (Addgene, #11151; 2 µg/µL), pCag-GFP (Addgene, #11150; 1 µg/µL), pCALNL-GFP (Addgene, #13770; 1 µg/µL), and pCag-Cre (Addgene, #13775; 4 ng/µL) (62 (link), 63 (link)). Plasmids were purified with a Maxiprep Endofree Kit (Macherey-Nagel) and diluted in 1x PBS. pCag-DsRed was added to the mix of electroporation as a control of efficiency. The DNA solution (with 0.05% Fast-green) was injected into the lateral ventricle of E15.5 embryos using pulled glass pipettes. The embryos were electroporated using tweezer-type electrodes (CUY650-5). Five square electric pulses were passed (40V, 50-ms interval cycle length, 950-ms interval pause). Electroporated animals, expressing DsRed, were selected using a fluorescent flashlight lamp (Nightsea, DFP-1).
8
Transfection Efficiency Quantification
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EJ5 reporter cells were grown in 6-cm dishes and co-transfected with 250 pmol of the indicated siRNA for 2 days and re-transfected with 250 pmol siRNA, 4 μg pCMV3xnlsI-SceI or phCMV-1 I-SceI (non-functional control) and 0.5 μg pCAG Ds-Red (Addgene) as a transfection control. After 72 h, cells were washed 2 × with PBS and analyzed using a FACSCantoII flow cytometer (BD Biosciences) and Flowing Software.
9
Lentiviral Cloning and Expression Protocols
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Lentiviral cloning and expression vectors pCDH-UbC-MCS-EF1-Hygro (CD615B-1) and pCDH-EF1-MCS-T2A-Puro (CD520A-1) were purchased from System Biosciences Inc. (SBI).
Human cDNA clones for LEPR transcript variant 1 (Catalog # HG10322-M), BBS1 (Catalog # HG10498-M) and BBS10 (Catalog # HG15095-G) were purchased from SBI. (63) .
Fluorescent expression vectors pEGFP-N3 (Clontech) and pCAG-DsRed (Addgene) were used in this study. Genes for GFP or RFP were cloned into the CD520/CD615 with BamHI and NotI digestion. CD520-RFP-LEPR was generated as described elsewhere (23) . 3xFLAG-BBS1 cDNA was PCR-amplified from the BBS1 cDNA plasmid using the following primers:
Human cDNA clones for LEPR transcript variant 1 (Catalog # HG10322-M), BBS1 (Catalog # HG10498-M) and BBS10 (Catalog # HG15095-G) were purchased from SBI. (63) .
Fluorescent expression vectors pEGFP-N3 (Clontech) and pCAG-DsRed (Addgene) were used in this study. Genes for GFP or RFP were cloned into the CD520/CD615 with BamHI and NotI digestion. CD520-RFP-LEPR was generated as described elsewhere (23) . 3xFLAG-BBS1 cDNA was PCR-amplified from the BBS1 cDNA plasmid using the following primers:
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