mPiezo2-CMV-Sport6
(Plasmid #81073)

• Purpose: mouse Piezo2
• Depositing Lab: Ardem Patapoutian
• Publication: Coste et al Nat Commun. 2015 May 26;6:7223. doi: 10.1038/ncomms8223.

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

Resource Information

• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

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

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.