Dear all,
I am now facing a problem. After ligation of a 12000 bp vector with 2000 bp gene fragment, no clones can be obtained after performing DH5alpha transformation?
Therefore, are there any suggestions? If DH5 alpha cant be used to clone such a large gene, what strain can I use?
Thank you.
It will be a bit more difficult with dh5 but is doable just use a higher amount of competent cells. For constructs of that size however neb10beta is recommended.
If you donot get any colony on the plate, it might be because the cells are not viable, try to prepare fresh cells.Hope so it will work
Yes you can do it but as others have mentioned transformation efficiency may be reduced. Generally electroporation is less size limited than chemical transformation so that might be a way to go, but you should still be able to get it via normal chemical transformation if your cells are highly competent.
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