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No colonies after self Circularization ligation - (Oct/23/2014 )



Im having a problem with my ligation and not sure how to proceed... My vector has two Pci I cut sites and i used it to cut out a fragment from my vector that i did not want there. I then used thermoscientific ligation kit to self circularize my vector. However after plating onto an Xgal IPTG induced amp plates, i either get no colonies or just light blue colonies. My vector is a pTZ-57RT plasmid with an insert, so shouldn't i only be getting white colonies because the beta galactosidase gene was already interrupted with my insert? did i need to do blue white screening for my self circularization experiment. also how could i get more colonies. My transformation positive control plate was fine so i don't think transformation efficiency was a problem. I used 20 ng of DNA the protocol said between 10-50 ng of linearised DNA.


Are you sure your digestions worked?


How did your control ligations look?


According to the maps/sequences I found on line PciI only cuts pTZ57R once (note not the pTZ57R/T, which should just stand for TA cloning, so sequence shouldn't be too different)... could this be the problem?


I think the idea is that Khaleesi had pTZ57R that somebody else had already put an insert in using that PciI site and Khaleesi wanted to remove that insert.


I'm assuming that you ran your sample on a gel after digesting and saw bands for the vector and the insert you were cutting out? Because if you cut out a band for just the vector and add ligase, that reaction should be crazy efficient.


You could try miniprepping liquid cultures from some of those light blue colonies and seeing if the plasmids are the right size for not having the insert.