double digest pcDNA(+) by XhoI&XbaI, uncompletely? - (Mar/15/2006 )
I digested pcDNA (+) by XhoI and XbaI, since these two enzyme sites are very close, CTCGAGTCTAGA, so I cannot say it's digested completely. But based on my ligation, I wanna insert a 3KB fragment which is digested by SalI and XbaI into pcDNA. I tried several times. The first time, I didn't get any clony. The second time, I got 9 clonies, but after one enzyme digestion and gel running to check the DNA, it's all 3kb band. How to explain it? The third time, I got 4 clonies. two of them are 3KB, two of them are 5kb. I follow the ration insert: vector=3:1. Now I am thinking that is because pcDNA cannot be digested completely. Is there any method to digest two close enzyme sites completely?
Thanks in advance.
You can do a three-step ligation in stead of the two step you are doing now.
This means that you look for a unique restriction site in the vector about 2/3 to the XhoI and XbaI site (in the circular plasmid map). Then you can enzyme X-XhoI and Enzyme x-XbaI. You can check if that has cut and isolate them from gel. Then do a ligation with the three fragments.
This means that you look for a unique restriction site in the vector about 2/3 to the XhoI and XbaI site (in the circular plasmid map). Then you can enzyme X-XhoI and Enzyme x-XbaI. You can check if that has cut and isolate them from gel. Then do a ligation with the three fragments.
Thanks for your suggestion!
It's a nice idea. But I am wondering if I can get the small fragment between enzyme X and XhoI. It's only about 20-30bp. which agarose percentage I have to choose for getting a so small fragment? And what's the optimal ration and time for ligation?
The pcDNA is about 5 kb right? I mean to isolate an enzyme X-XhoI fragment of about 2kb and an enzymeX-XbaI fragment of about 3kb, so choose enzyme X that it lays 2 kb from XhoI and 3 kb from XbaI (or the other way around). So you don't have to isolate such as small fragment, it is possible on an acrylamide gel, but that's not very easy.
I would add these two fragments in the same ratio and the insert as 3:1. The ligation time depends on the ligase, read the manual whats the best, o/n 16 or 4C or 1-2 hours RT or 37C.