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Species: Mus musculus
Genetic Insert: Smad7
Vector Backbone Description: Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_80893 Copy
Species: Mus musculus
Genetic Insert: ALK6
Vector Backbone Description: Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26701726
Proper citation: RRID:Addgene_80884 Copy
Species: Mus musculus
Genetic Insert: ALK6
Vector Backbone Description: Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26701726
Proper citation: RRID:Addgene_80882 Copy
Species: Mus musculus
Genetic Insert: Gsdmd
Vector Backbone Description: Backbone Marker:Sigma; Vector Backbone:pFlag-CMV-4; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:27383986
Proper citation: RRID:Addgene_80950 Copy
Species: Mus musculus
Genetic Insert: TET1
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:4008; Vector Backbone:pACYCDuet-1; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:26932196
Proper citation: RRID:Addgene_81056 Copy
Species: Mus musculus
Genetic Insert: TET1
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:3780; Vector Backbone:pCDF-Duet-1; Vector Types:Bacterial Expression; Bacterial Resistance:Streptomycin
Defining Citation: PMID:26932196
Proper citation: RRID:Addgene_81053 Copy
Species: Mus musculus
Genetic Insert: TET1
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:3780; Vector Backbone:pCDF-Duet-1; Vector Types:Bacterial Expression; Bacterial Resistance:Streptomycin
Defining Citation: PMID:26932196
Proper citation: RRID:Addgene_81054 Copy
Species: Mus musculus
Genetic Insert: Raptor shRNA
Vector Backbone Description: Backbone Marker:Jacks Lab; Backbone Size:7566; Vector Backbone:pSico; Vector Types:Mammalian Expression, Lentiviral, RNAi, Cre/Lox; Bacterial Resistance:Ampicillin
Defining Citation: PMID:24139800
Comments: pSico allows for conditional (Cre-Lox), stable expression of shRaptor. Addition of Cre excises GFP and allows for shRaptor expression.
Proper citation: RRID:Addgene_81086 Copy
Species: Mus musculus
Genetic Insert: Piezo2 (please see depositor comments below)
Vector Backbone Description: Vector Backbone:pCMV-Sport6; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26008989
Comments: Please note- when compared to the Piezo2 reference sequence NM_001039485.4, the insert in this plasmid contains mutation D1896E and deletion of residues V1897 and E1898. Piezo2 was amplified out of mouse DRGs. The variant that is present in DRGs when it was cloned is missing 6 base pairs. Here is the link to the original manuscript—the reported protein sequence is in the supplementary data. The plasmid functions as described. http://science.sciencemag.org/content/330/6000/55.full
The depositor recommends the following protocol for researchers having issues growing or isolating plasmid 81073 (mPiezo2-CMV-Sport6):
For Piezo2- inoculate culture from glycerol stock into 4-5mls of LB media. Grow the culture at 37 deg for 4-5 hrs and then transfer the culture to larger volumes of media (about 100-150mls) and grow overnight at 37 deg. Usual maxi prep yields are low in the range of 50-100ng/ul. We digest verify the preps by digesting with KpnI and NotI to give 8.2kb insert and 4.4kb vector bands or with KpnI and SpeI to give 2 bands with sizes 8.6 kb and 4.2 kb.
When handling glycerol stocks for Piezo2, always re-streak and pick single clones. Never inoculate the glycerol stock into an overnight culture. Our lab has had issues with clones that grow with amp resistance but after digestion, show a prominent ~4 kb single band. After mini-prep or maxi-prep, always confirm the DNA product by restriction digest. We digest with KpnI and SpeI and this leads to 2 bands with sizes 8.6 kb and 4.2 kb. We digest verify every time we maxi prep. For our maxi preps, we notice that our yields are low - sometimes ~ 50-100 ng/ul when eluting in 1 ml from ~150 ml of culture.
Alternatively, transform your DNA into XL-10 gold cells. Plate and grow for 3-4 days at 25C. Select single colonies and inoculate 200-300 mL of media. Grow with shaking at 25C for 3-4 days. Maxiprep the culture as normal, digest DNA to verify as above.
Proper citation: RRID:Addgene_81073 Copy
Species: Mus musculus
Genetic Insert: CRT
Vector Backbone Description: Backbone Marker:Steyger lab; Vector Backbone:GFP-pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21785162
Proper citation: RRID:Addgene_80978 Copy
Species: Mus musculus
Genetic Insert: TDG
Vector Backbone Description: Backbone Marker:New England Biolabs; Backbone Size:6550; Vector Backbone:pTXB3; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26932196
Proper citation: RRID:Addgene_81048 Copy
Species: Mus musculus
Genetic Insert: TDG
Vector Backbone Description: Backbone Marker:New England Biolabs; Backbone Size:6550; Vector Backbone:pTXB3; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26932196
Proper citation: RRID:Addgene_81049 Copy
Species: Mus musculus
Genetic Insert: 4EBP1 F113A
Vector Backbone Description: Backbone Size:4802; Vector Backbone:pCAGGS; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:24139800
Comments: These plasmids were also used in the following publication "Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration. Lin TV, Hsieh L, Kimura T, Malone TJ, Bordey A. Proc Natl Acad Sci U S A. 2016 Oct 4;113(40):11330-11335."
Proper citation: RRID:Addgene_81122 Copy
Species: Mus musculus
Genetic Insert: 4E-BP2 shRNA
Vector Backbone Description: Backbone Marker:Jacks Lab (MIT); Backbone Size:7567; Vector Backbone:pSicoR; Vector Types:Mammalian Expression, Lentiviral, RNAi, Cre/Lox; Bacterial Resistance:Ampicillin
Defining Citation: PMID:24139800
Comments: pSicoR allows for expression of the 4E-BP2 shRNA in the absence of Cre. Expression of Cre turns off shRNA expression. These plasmids were also used in the following publication "Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration. Lin TV, Hsieh L, Kimura T, Malone TJ, Bordey A. Proc Natl Acad Sci U S A. 2016 Oct 4;113(40):11330-11335."
Proper citation: RRID:Addgene_81121 Copy
Species: Mus musculus
Genetic Insert: SKP2
Vector Backbone Description: Backbone Marker:Promega; Vector Backbone:pGL2-basic; Vector Types:Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:27309807
Proper citation: RRID:Addgene_81119 Copy
Species: Mus musculus
Genetic Insert: immunoglobulin heavy constant mu
Vector Backbone Description: Backbone Marker:Invivogen; Backbone Size:4852; Vector Backbone:pFUSEss-CHIg-mM; Vector Types:Mammalian Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:29323348
Proper citation: RRID:Addgene_91729 Copy
Species: Mus musculus
Genetic Insert: immunoglobulin heavy constant mu
Vector Backbone Description: Backbone Marker:Invivogen; Backbone Size:4852; Vector Backbone:pFUSEss-CHIg-mM; Vector Types:Mammalian Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:29323348
Proper citation: RRID:Addgene_91747 Copy
Species: Mus musculus
Genetic Insert: immunoglobulin heavy constant mu
Vector Backbone Description: Backbone Marker:Invivogen; Backbone Size:4852; Vector Backbone:pFUSEss-CHIg-mM; Vector Types:Mammalian Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:29323348
Proper citation: RRID:Addgene_91748 Copy
Species: Mus musculus
Genetic Insert: immunoglobulin heavy constant mu
Vector Backbone Description: Backbone Marker:Invivogen; Backbone Size:4852; Vector Backbone:pFUSEss-CHIg-mM; Vector Types:Mammalian Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:29323348
Proper citation: RRID:Addgene_91745 Copy
Species: Mus musculus
Genetic Insert: immunoglobulin heavy constant mu
Vector Backbone Description: Backbone Marker:Invivogen; Backbone Size:4852; Vector Backbone:pFUSEss-CHIg-mM; Vector Types:Mammalian Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:29323348
Proper citation: RRID:Addgene_91746 Copy
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