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How to solve the problem of toxic ligation? - Are there any other competent cells better than DH5a dealing with toxi (Jan/03/2007 )

Hi, there:
I just joined this site today. Maybe you've already discussed the topic before, but can anybody copy and paste your answers if you've done this topic before since I still don't know where to look for my answers? Thanks.
Here is my problem. I have been trying to make a construct to make conditional knockout mice. I am on the last step to put two arms (with the neo gene between and loxP at certain place) together, but it just doesn't work!!! Before this, I tried to put two arm together first (with loxP) and it didn't work. All my other ligations/transformations for the knockout mice worked at the same time except this one. My arms usually are around 3.5 to 5 kb, generally speaking. Is this so called "toxic ligation"? How to solve this problem? I use DH5 alpha. Are there any other competent cells which can take toxic ligation?
Thank you very much for your helps.
Happy new year, everybody.


could use a strain which has low recombination rates... DH10 cells for examples

you can also grow your cells at a lower temperature - 30 Celsius. That sometimes helps with this kinds of ligation problem.

And make sure your cells aren't expressing Cre tongue.gif


Stratagene makes a bunch of different competent cells for problem vectors. I successfully used the SURE cells with a highly repetitive insert that was excised out by normal DH5-alpha cells.


Stratagene's ABLE cells sometimes work for toxic constructs as they reduce the copy number right down, I think they sell a 'difficult construct pack' or something that has both SURE2 and ABLE cells in it, perhaps you could try that?