PurposeTemplate DNA for in vitro transcription using T7 polymerase
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||50563||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 2710
- Total vector size (bp) 4016
Vector typecloning vector
Growth in Bacteria
- Promoter lac
- Cloning method Restriction Enzyme
- 5′ cloning site unknown (unknown if destroyed)
- 3′ cloning site unknown (unknown if destroyed)
- 5′ sequencing primer T7
- 3′ sequencing primer M13R (Common Sequencing Primers)
A portion of this plasmid was derived from a plasmid made byRluc gene derives from psiCheck2 from Promega.
Terms and Licenses
Plasmid contains 8 copies of the let-7 target site, a defined A114 sequence followed by an EcoT22I site and a 40-bp arbitrary sequence all downstream of Renilla lucferase.
Unstable when transformed and so any retransformation must be checked by digest or sequencing before use. In strain MV1184, it is stable as a glycerol stock. Please note that the Rluc coding sequence contains an EcoT22I site. To prepare the T7 transcription template for A114 RNA (without N40), the plasmid should be first linearized by EcoRI upstream of the T7 promoter sequence, followed by a partial digestion by EcoT22I. The DNA fragment terminating immediately downstream of the A114 sequence should then be gel purified. For A114-N40 RNA, the plasmid should be digested by HindIII downstream of the N40 sequence.
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:pUC57-Rluc-let-7-A114-N40 was a gift from Yukihide Tomari (Addgene plasmid # 50563 ; http://n2t.net/addgene:50563 ; RRID:Addgene_50563)
For your References section:PABP is not essential for microRNA-mediated translational repression and deadenylation in vitro. Fukaya T, Tomari Y. EMBO J. 2011 Nov 25;30(24):4998-5009. doi: 10.1038/emboj.2011.426. 10.1038/emboj.2011.426 PubMed 22117217