Trouble cloning a 8.5 kb fragment into a pTRE vector - (Jun/16/2005 )
please help me with my problem.
I've got a linear 8.5kb fragment, on the 5' end a unphosphorylated MluI site, on the 3' end a phosphorylated NheI site - this is just due to the quite complicated way I produced this fragment. The amount is not very much (100ng) and that is all I got. I want to clone this fragment in a pTRE tight vector (2.6kb) and in the past it turned out that this could be tricky.
Is there a easy way to subclone this fragment into another vector. This would be a comfortable situation for production of high amounts of this fragment - Since the generation of this fragment always takes two days and the final cloning of this fragment into the pTRE vector(final aim) always failed. Some people say, you would have to bear in mind that subcloning of a 8.5kb fragment into a high copy plasmid (like pGEM Yeasy, Bluescribt etc) could make trouble, since cells simply would dye!
Am I driving in a dead end or does anybody know a way out?
P.S. Help will be highly appreciated
I'm not going to touch this one.. anyone else willing to give it a try?
Sorry I coudn't help.
it might help to increase the concentration of purity of your vector backbone. In my experience, you can clone almost every insert when your backbone is perfectly prepared. For the low-copy pTre-tight, look at this post
8.5 kB is large, but works when you use the right E. coli strain. I had good experience using XL10Gold for large insert-clonings;