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URL: http://www.addgene.org/21189

Proper Citation: RRID:Addgene_21189

Insert Name: GateWay conversion cassette

Bacterial Resistance: Chloramphenicol and Ampicillin

Defining Citation: PMID:19014667

Vector Backbone Description: Backbone Size:14820; Vector Backbone:pRosa26-PA; Vector Types:Mammalian Expression; Bacterial Resistance:Chloramphenicol and Ampicillin

Comments: pRosa26-DEST was made by inserting a GateWay conversion cassette into the XhoI site of pBigT, followed by transfer of PacI / AscI fragment of the resulting vector to the pRosa26-PA vector (see http://www.biomedcentral.com/1471-213X/1/4 for details on these vectors). The resulting vector resembles the construct that was used for the R26R Cre reporter mouse made by Phil Soriano in which the expression of lacZ is coupled to the endogenous Rosa26 locus via a splice acceptor site, but only after removal of the lox-STOP-lox cassette (which is actually the neo-R cassette used during the targeting). The only thing changed from the pBigT vectors and the R26R reporter is the cloning strategy, so the resulting knock-in should be as good as these systems, and as good (or as bad) as the use of the Rosa26 locus in any situation. Stbl3 cells are advisable to use after users have done the LR recombination reaction. See http://www.hgu.mrc.ac.uk/people/n.hastie_rosa.html for more information and protocols for this plasmid. Further advice from the depositing lab: "Grow single colonies in Amp + Cam at 30 C, definitively not 37. Including the Cam should ensure that spontaneous recombinants that loose the Gateway cassette don't survive... - Do minipreps and test with the double digest PacI / AscI. This should give 4.9 kb + 9.8 kb. If this is okay, the vector should be okay" Please note that there are discrepancies between Addgene's quality control sequence and the theoretical sequence supplied by the depositing lab. These discrepancies should not affect the function of the construct.

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