Partial Digestion and Religation - Partial Digestion to eliminate a restriction site (Jul/24/2007 )
I'm trying to eliminate an EcoRI site in a plasmid localized at 5' of my insert of interest cause I want to use an internal EcoRI for cloning a smaller fragment.
I've made a partial digestion with EcoRI, select the band that has been cut only once, blunt the ends with T4 DNA pol and religate itself. After transforming, I'm checking the colonies, and more than 80% of them has lost the internal EcoRI site (which I don't want to lose). The rest of the colonies are a result of religation of molecules cut with restriction enzyme's "star" activity, and they're not usefull.
I assume that this is because the digestion of one of the sites is favoured regarding the other (correct me if I'm wrong). Does anybody know how to change the digestion conditions to favour the restriction of the least probable site? Or do I have to follow trying to find the good colony?
Star activity? Hmm... out of curiousity could you write out your digesting mixture? Such high levels of star activity is kind of strange. And how many colonies did you check?
Perhaps changing the buffer your digest is conducted in might help.
Still, if your plasmid is small enough, you might consider PCR amplifying the vector. Not the best solution in this case, but could be done. A similar method is site directed mutagenesis.