tricky digestion for cloning gurus - (Jan/24/2006 )
Yet another ligation problem involving XhoI.
The evidence is mounting people.
There is nothing wrong with Xho I. It's one of the better ones out there (like Nco I).
I am with pBluescript, but I would qualify it...I have had good luck with single-cut XhoI clonings, they have worked like a charm. I have struggled mightily with XhoI / enzymeX clonings. these should in fact be easier....two different sticky ends, you can't go wrong, you should barely have to add ligase, right? but it seems with XhoI they are trickier
I am wondering if someone screwed up in the methods section and it was a different pET vector after all?
maybe...or they were just very lucky! sometimes at the first try you get what you want and if you need to repeat it, you just can´t get on with it!!
you can always do mutagenesis to add or modify the restriction sites. usually works well. with complementary primers with the desired mutations pfu and DpnI you get the job done in 2-3 days.