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Species: Mus musculus
Genetic Insert: Pip5k1c
Vector Backbone Description: Vector Backbone:pEGFP; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:27447049
Comments: Three modifications have been made to PIP5K1g (NM008844): R445E and R446E mutations to favour cytosolic distribution and truncation of amino acids 636-661 to prevent plasma membrane localisation.
Proper citation: RRID:Addgene_79569 Copy
Species: Mus musculus
Genetic Insert: syntaxin 1A
Vector Backbone Description: Vector Backbone:pEGFP; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:22847441
Comments: This plasmid contains the C-terminal region (polybasic stretch and transmembrane domain; amino acids 249-288) from human Syntaxin 1A (NM_004603.3).
Proper citation: RRID:Addgene_79571 Copy
Species: Mus musculus
Genetic Insert: Trex2
Vector Backbone Description: Vector Backbone:pCAGGS; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19834534
Proper citation: RRID:Addgene_79246 Copy
Species: Mus musculus
Genetic Insert: Paxillin
Vector Backbone Description: Vector Backbone:pEGFP; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_80029 Copy
Species: Mus musculus
Genetic Insert: Talin
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23468527
Proper citation: RRID:Addgene_80026 Copy
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: 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: 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: 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: 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: mouse NFL
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4242; Vector Backbone:pEGFP-C1 with EGFP sequence removed; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:17285620
Comments: Plasmid first described in Yan et al. (2007), "The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons", Cell Motility and the Cytoskeleton 64:299–309.
Mouse NFL cDNA sequence deposited in Genbank (Accession number DQ201635).
Proper citation: RRID:Addgene_83127 Copy
Species: Mus musculus
Genetic Insert: mouse NFM
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4047; Vector Backbone:pEGFP-C1 with EGFP sequence removed; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:17285620
Comments: Plasmid first described in Yan et al. (2007), "The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons", Cell Motility and the Cytoskeleton 64:299–309.
Mouse NFM cDNA sequence deposited in Genbank (Accession number DQ201636).
Proper citation: RRID:Addgene_83126 Copy
Species: Mus musculus
Genetic Insert: sgRNA targeting mouse CD90.2
Vector Backbone Description: Backbone Marker:Feng Zhang (Addgene plasmid# 48138); Backbone Size:9300; Vector Backbone:pX458; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29445007
Comments: Plasmid co-expresses Cas9 from S. pyogenes with 2A-EGFP, as part of the pX458 backbone. These plasmids were previously associated with the following preprint: Highly efficient and versatile plasmid-based gene editing in primary T cells bioRxiv. 13 January 2018. doi:10.1101/247544
Proper citation: RRID:Addgene_82670 Copy
Species: Mus musculus
Genetic Insert: sgRNA targeting mouse CD45.2 region 1
Vector Backbone Description: Backbone Marker:Feng Zhang (Addgene plasmid# 48138); Backbone Size:9300; Vector Backbone:pX458; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29445007
Comments: Plasmid co-expresses Cas9 from S. pyogenes with 2A-EGFP, as part of the pX458 backbone. These plasmids were previously associated with the following preprint: Highly efficient and versatile plasmid-based gene editing in primary T cells bioRxiv. 13 January 2018. doi:10.1101/247544
Proper citation: RRID:Addgene_82672 Copy
Species: Mus musculus
Genetic Insert: 160bp genomic DNA sequence from murine CD90.1
Vector Backbone Description: Backbone Marker:Genscript; Backbone Size:2710; Vector Backbone:pUC57; Vector Types:HDR template for gene editing; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29445007
Comments: This HDR template can be used in conjunction with sgRNA CD90.2 (Addgene ID 82670 or Addgene ID 82671). In the template we introduced a PAM mutation. These plasmids were previously associated with the following preprint: Highly efficient and versatile plasmid-based gene editing in primary T cells bioRxiv. 13 January 2018. doi:10.1101/247544
Proper citation: RRID:Addgene_82666 Copy
Species: Mus musculus
Genetic Insert: USH1C
Vector Backbone Description: Backbone Marker:Vector Builder ; Backbone Size:3855; Vector Backbone:pRP[Exp]-CMV; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_83202 Copy
Species: Mus musculus
Genetic Insert: NES mRNA capping enzyme
Vector Backbone Description: Backbone Size:7224; Vector Backbone:pcDNA3-myc-mCE(K294A); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19223470
Proper citation: RRID:Addgene_82464 Copy
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