I am trying to find out what are the problems in my ligations.
The first test: linearized Plasmid+ AP+ ligase buffer, after transformation, it gives a lot of colonies !
The second test: linearized Plasmid+ AP+ ligase buffer + Ligase, after transformation, it gives 0 colony !
I don't know what can be the problem because if the first test can give colonies without ligase, how come the second test can give 0 colony even ligase added. please help me ! Thank you !
did you gel purify the linear cut form of the plasmid? if not...this is the main reason explaining the high background
as a general guideline...always gel purify your linear plasmid...ideally, run a well resolved band (long migration , low voltage) , cut out from the gel and column purify, analyse and aliquot to assess the quantity and quality...
Didier Fesquet Thank you Sir for your answer.
I purified the linear cut and inactivated residues from AP at 800 C, 10 mins before I did ligations
You are correct that the results appear reversed. First, I presume you are adding an insert as well? Because if everything was correct you should not get colonies from either test. Is this the exact same preparation of linearized plasmid? If you just repeated the digestion and purification twice, it could be that one purification failed.
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