|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||102646||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerMarkus Ralser (Addgene plasmid # 19407), Clontech = pTRE-T2
Vector typeMammalian Expression, RNAi
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
Terms and Licenses
- Not Available to Industry
In fusion cloning was used to insert a synthetic DNA template containing the mIR cassette from the pCDNA 6.2-GW/miR into the EcoRI and XbaI site of pTRE-T2-Tight. I engineered two BsaI sites into the miR cassette so users can digest the vector with BsaI for directional cloning of Blockit microRNAs using the Block-IT RNAi designer engine from Invitrogen. To make the BsaI sites unique I mutated out Bsa I sites in the original pTRE-T2 Tight vector and also removed XhoI and XbaI sites by site-directed mutagenesis from the pTRE-T2 vector and the corresponding puromycin/hygromycin resistance genes. I also dropped in a unique NheI and EagI restriction site between the ampicillin gene and the pMB101 origin of replication sequence so that the puromycin and hygromin genes could be placed into the pTRE-T2-miR vector with the infusion cloning method. Investigators can remove the miR cassette from the pTRE-T2 puromycin/hygromycin vector and using cloning methods to insert any cDNA of interest into the site for doxycline-inducible expression of the target cDNA. A single vector can be utilized from the production of tetracycline inducible miRNAs or cDNAs.
For puromycin use .25 micrograms/ml to .50 micrograms/ml to get clones after a couple of weeks. Growth response curve tested in 293HEK cells. Suggest methylation deficient cells from NEB to alleviate methylation sensitive restriction sites like Xba I. However not recommended for long-term propagation and storage.
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:pTRE-T2-miR-PURO was a gift from Michael E. Wright (Addgene plasmid # 102646 ; http://n2t.net/addgene:102646 ; RRID:Addgene_102646)