I have been struggling with ligation for long fragment(7kb+11kb).The vector is 11kb and was digested by ASCⅠ(a stick end) and NruⅠ(a blunt end).After digestion,phosphatase was added into the reaction. The insert is 7kb and was digested by the same restriction endonuclease.However,i cannot get a transformant which i need and the produce has strange fragment by agarose gel electrophoresis under ASCⅠ and NruⅠ.Why the strange fragment appeared ?How can i do?My teacher said that i did a excessive dephosphorylation by phosphatase,but i do't understand.
Hi Zhang can you be more specific about the sizeof the strange fragments in your products. Also for any ligation it is good to run an empty vector ligation for control. Did you run this control and does it produce colonies?
I have not heard of too much phosphatase, but am thinking you need to prevent vector self ligation and optimize vector to insert ratio.
A couple of suggestions: one is also to use either infusion or NEB's gibson assembly
I usually do restriction free cloning, I have had much more success with it, even with large fragments. Look at http://www.rf-cloning.org
Since your construct is quite large you don't expect a high efficiency. I guess that you have checked your vector and insert on a gel. If you don't want to try another approach (I like Gibson assembly), I recommend to ommit the phosphatase step. If both of your enzymes cut you won't get religation and without dephosphorylation you expect more positive clones.
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