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Species: Mus musculus
Genetic Insert: Type I procollagen α1 chain
Vector Backbone Description: Backbone Size:5300; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30287488
Comments: These mouse type I procollagen plasmids were designed for immortalized and primary murine osteoblasts and fibroblasts. Their expression in other cells might cause significant disruption of assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in osteoblasts and fibroblasts a fraction of transfected chains, particularly proα2, might be trafficked/degraded as monomers instead of being integrated into procollagen heterotrimers. In our experience, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12 h after transfection. Fluorescent molecules should be incorporated into fibers even when the transfected chains contain the N-propeptide cleavage site, because the cleavage is highly conformation sensitive and always incomplete.
We recommend following these guidelines when using the plasmids
1) Only mouse cells that have high expression of endogenous type I procollagen are suitable for studies of physiologically-relevant processes with these constructs.
2) At least 100 μM ascorbic acid at and after transfection is required to ensure proper procollagen folding (for some cells, preincubation with ascorbic acid before transfection might be needed).
3) Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid excessive accumulation of aggregates and cell malfunction caused by increased procollagen synthesis.
5) Lack of extracellular fluorescent fibers 12-24 h after transfection indicates improper procollagen synthesis/trafficking or cellular malfunction.
6) Excessive expression of transfected chains might cause rapid accumulation of large procollagen aggregates in the ER, resulting in cellular malfunction and death.
Contact information: Shakib Omari ([email protected]), Sergey Leikin ([email protected]).
Proper citation: RRID:Addgene_119844 Copy
Species: Mus musculus
Genetic Insert: Type I procollagen α1 chain
Vector Backbone Description: Backbone Size:5300; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30287488
Comments: These mouse type I procollagen plasmids were designed for immortalized and primary murine osteoblasts and fibroblasts. Their expression in other cells might cause significant disruption of assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in osteoblasts and fibroblasts a fraction of transfected chains, particularly proα2, might be trafficked/degraded as monomers instead of being integrated into procollagen heterotrimers. In our experience, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12 h after transfection. Fluorescent molecules should be incorporated into fibers even when the transfected chains contain the N-propeptide cleavage site, because the cleavage is highly conformation sensitive and always incomplete.
We recommend following these guidelines when using the plasmids
1) Only mouse cells that have high expression of endogenous type I procollagen are suitable for studies of physiologically-relevant processes with these constructs.
2) At least 100 μM ascorbic acid at and after transfection is required to ensure proper procollagen folding (for some cells, preincubation with ascorbic acid before transfection might be needed).
3) Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid excessive accumulation of aggregates and cell malfunction caused by increased procollagen synthesis.
5) Lack of extracellular fluorescent fibers 12-24 h after transfection indicates improper procollagen synthesis/trafficking or cellular malfunction.
6) Excessive expression of transfected chains might cause rapid accumulation of large procollagen aggregates in the ER, resulting in cellular malfunction and death.
Contact information: Shakib Omari ([email protected]), Sergey Leikin ([email protected]).
Proper citation: RRID:Addgene_119842 Copy
Species: Mus musculus
Genetic Insert: Type I procollagen α2 chain
Vector Backbone Description: Backbone Size:5400; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30287488
Comments: These mouse type I procollagen plasmids were designed for immortalized and primary murine osteoblasts and fibroblasts. Their expression in other cells might cause significant disruption of assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in osteoblasts and fibroblasts a fraction of transfected chains, particularly proα2, might be trafficked/degraded as monomers instead of being integrated into procollagen heterotrimers. In our experience, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12 h after transfection. Fluorescent molecules should be incorporated into fibers even when the transfected chains contain the N-propeptide cleavage site, because the cleavage is highly conformation sensitive and always incomplete.
We recommend following these guidelines when using the plasmids
1) Only mouse cells that have high expression of endogenous type I procollagen are suitable for studies of physiologically-relevant processes with these constructs.
2) At least 100 μM ascorbic acid at and after transfection is required to ensure proper procollagen folding (for some cells, preincubation with ascorbic acid before transfection might be needed).
3) Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid excessive accumulation of aggregates and cell malfunction caused by increased procollagen synthesis.
5) Lack of extracellular fluorescent fibers 12-24 h after transfection indicates improper procollagen synthesis/trafficking or cellular malfunction.
6) Excessive expression of transfected chains might cause rapid accumulation of large procollagen aggregates in the ER, resulting in cellular malfunction and death.
Contact information: Shakib Omari ([email protected]), Sergey Leikin ([email protected]).
Proper citation: RRID:Addgene_119840 Copy
Species: Mus musculus
Genetic Insert: myelocytomatosis oncogene-F2A-Kruppel-like factor 4-T2A-POU domain, class 5, transcription factor 1-E2A-SRY-box containing gene
Vector Backbone Description: Backbone Size:6397; Vector Backbone:pCAG-ires-EGFP; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:19252477
Proper citation: RRID:Addgene_20865 Copy
Species: Mus musculus
Genetic Insert: Notch 3 Intracellular Domain
Vector Backbone Description: Backbone Marker:Sigma; Backbone Size:4700; Vector Backbone:p3XFLAG-CMV-7; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16365048
Comments: All NICD plasmids were cloned into the p3XFLAG-CMV-7(TM) expression vector (Sigma). NICD3 was cloned into XbaI/BamHI sites. NICD3 (Met-1664 to Ala-2318 of full-length mouse N3) was amplified from the N3 plasmid (gift from Dr. U. Lendahl) with 5XbaN3 (5'-GCTCTAGAATGGTTGCCAGGCGAAAGCGAG-3') and 3BamN3
(5'-GCGGATCCCTAGGCCATCACCTGCCTCTTGGG-3') primers.
Proper citation: RRID:Addgene_20185 Copy
Species: Mus musculus
Genetic Insert: Notch 2 intracellular domain
Vector Backbone Description: Backbone Marker:Sigma; Backbone Size:4700; Vector Backbone:p3XFLAG-CMV-7; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16365048
Comments: All NICD plasmids were cloned into the p3XFLAG-CMV-7(TM) expression vector (Sigma). NICD2 was cloned into XbaI/BamHI sites. NICD2 (Met-1699 to Ala-2470 of full-length mouse N2) was amplified from the N2 plasmid (gift from Dr. Y. Hamada) using primers 5XbaN2 (5'-GCTCTAGAATGGCCAAGCGGAAGCGCAAG-3') and 3BamN2
(5'-GCGGATCCCTATGCATACACCTGCATGTTGCTG-3').
Please note that Addgene sequencing results confirm the updated domain numbering to be Met 1701-Ala 2473.
Proper citation: RRID:Addgene_20184 Copy
Species: Mus musculus
Genetic Insert: C-Fluc
Vector Backbone Description: Backbone Marker:Ohlfest Lab; Backbone Size:6082; Vector Backbone:colE1/ampR; Vector Types:Transposon; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19147555
Proper citation: RRID:Addgene_20203 Copy
Species: Mus musculus
Genetic Insert: pDEST27-Vinculin
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:8123; Vector Backbone:pDEST27; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19075109
Proper citation: RRID:Addgene_20144 Copy
Species: Mus musculus
Genetic Insert: pCMV5 Flag p38 alpha
Vector Backbone Description: Backbone Marker:Dr. David Russell, UTSW; Backbone Size:4657; Vector Backbone:pCMV5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:7535770
Proper citation: RRID:Addgene_20786 Copy
Species: Mus musculus
Genetic Insert: pDCNA3 Flag p38 delta
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5500; Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:10716930
Proper citation: RRID:Addgene_20785 Copy
Species: Mus musculus
Genetic Insert: beta-transducin repeat containing (BTRC)
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5486; Vector Backbone:pcDNA3-myc3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11245432
Comments: *possible Y377I mutation in stock. This mutation is not known to affect function.
Proper citation: RRID:Addgene_20718 Copy
Species: Mus musculus
Genetic Insert: Sirt6
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4700; Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:15795229
Proper citation: RRID:Addgene_20275 Copy
Species: Mus musculus
Genetic Insert: CFP-FKBP-p85
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4733; Vector Backbone:ECFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:16990515
Proper citation: RRID:Addgene_20159 Copy
Species: Mus musculus
Genetic Insert: mIL-17
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:4242; Vector Backbone:pGL4.10; Vector Types:Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18849990
Proper citation: RRID:Addgene_20127 Copy
Species: Mus musculus
Genetic Insert: p21 3'UTR site 1 and 2 mutation
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5256; Vector Backbone:pGL3-Control; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18978791
Proper citation: RRID:Addgene_20880 Copy
Species: Mus musculus
Genetic Insert: Nfkb2
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5400; Vector Backbone:cFlag pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: See cFlag pcDNA3 plasmid for backbone information. 5' insert restriction site flanked by kozak/start site (CCACCATG).
Proper citation: RRID:Addgene_20019 Copy
Species: Mus musculus
Genetic Insert: RelB
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5400; Vector Backbone:cFlag pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: See cFlag pcDNA3 plasmid for backbone information. 5' insert restriction site flanked by kozak/start site (CCACCATG).
Proper citation: RRID:Addgene_20017 Copy
Species: Mus musculus
Genetic Insert: RelA
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5400; Vector Backbone:cFlag pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16166378
Comments: See cFlag pcDNA plasmid for backbone information. 5' insert restriction site flanked by kozak/start site (CCACCATG).
Proper citation: RRID:Addgene_20012 Copy
Species: Mus musculus
Genetic Insert: RelA
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5400; Vector Backbone:cFlag pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: See cFlag pcDNA3 plasmid for backbone information. 5' insert restriction site flanked by kozak/start site (CCACCATG).
Proper citation: RRID:Addgene_20015 Copy
Species: Mus musculus
Genetic Insert: Inhibitor of DNA binding 1
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:19896442
Proper citation: RRID:Addgene_20964 Copy
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