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mPiezo2-CMV-Sport6
(Plasmid #81073)

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This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 81073 Standard format: Plasmid sent in bacteria as agar stab 1 $94
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DNA 81073-D50 50 μg of DNA in Tris buffer $495
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DNA 81073-D100 100 μg of DNA in Tris buffer $585
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Backbone

  • Vector backbone
    pCMV-Sport6
  • Total vector size (bp) 12829
  • Vector type
    Mammalian Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    Room Temperature
  • Growth Strain(s)
    XL10 Gold
  • Growth instructions
    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.
  • Copy number
    Low Copy

Gene/Insert

  • Gene/Insert name
    Piezo2 (please see depositor comments below)
  • Species
    M. musculus (mouse)
  • Insert Size (bp)
    8475
  • Entrez Gene
    Piezo2 (a.k.a. 5930434P17, 9030411M15Rik, 9430028L06Rik, Fam38b, Fam38b2)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site KpnI (not destroyed)
  • 3′ cloning site NotI (not destroyed)
  • 5′ sequencing primer CGC AAA TGG GCG GTA GGC GTG
  • (Common Sequencing Primers)

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry

Trademarks:

  • Zeocin® is an InvivoGen trademark.

Depositor 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.

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

  • Amount 50 μg
  • Concentration 1 μg/μL
  • Pricing $495 USD
  • Storage 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

  • Amount 100 μg
  • Concentration 1 μg/μL
  • Pricing $585 USD
  • Storage 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    mPiezo2-CMV-Sport6 was a gift from Ardem Patapoutian (Addgene plasmid # 81073 ; http://n2t.net/addgene:81073 ; RRID:Addgene_81073)

  • For your References section:

    Piezo1 ion channel pore properties are dictated by C-terminal region. Coste B, Murthy SE, Mathur J, Schmidt M, Mechioukhi Y, Delmas P, Patapoutian A. Nat Commun. 2015 May 26;6:7223. doi: 10.1038/ncomms8223. 10.1038/ncomms8223 PubMed 26008989
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