Change pBI121 promoter - (Aug/10/2007 )

firstly, it is possible that your vector is not totally digested. And some vector molecules which were cut by only one enzyme was pull down with your full cut vector. It is also possible that you have some denatured plasmid pulled down with your cut vector. Denatured plasmid (which is produced by the cell lysis process) is resistent to digestion but will transform fine. The only way around this is to gel purify your vector slowly over a very large area (very big well). Thus allowing better separation. You can also gel purify your vector twice.

Double insert ligatio is not a concern in this situation. The probably is low that the vector will pick up two inserts as your ends are incompatible for a double insert ligation. In any case, it is not possible to eliminate the production of insert dimer production. The only way is to increase the concentration of vector, so that the insert is more likely to ligate to a vector molecule then to it self.

Perhaps I would clarify, a 1:3 ratio is a ratio in mols. not the amount of DNA. The vector is large (thus staining strongly) but how many mols are there? How much is being ligated here? I would suggest increasing the amount of DNA in the ligation mix both insert and vector.

Hmm... as for you ligation gel... Do you not seen any bands larger then your vector?

THe DH5 alpha cells, where do they comefrom and what transformation method are you using?

And finally how many colonies are you screening? 48? or 72?

Finally, before I forget. Did you dephosphorylate your vector? Over dephosphorylation can damage the vector's overhangs, rendering it unligatable.