Cloning Related Problem - Cloning in pBSK (Jul/22/2007 )
I am trying to clone a FL-cDNA (which is also in a vector pCMVFL3) in to the vector pBSK. The cloning is directional one and the enzymes selected are Eco RI and Hind III. After the digestion i hv got the desired fragment and then gel eluted it,ligated it using T4 DNA ligase. Alsa! i am not able to get a single transformed colony on selection plate.Ligase is working fine as it has been checked using the control (Single digested plasmid pBSK).
Please suggest me something where i am commiting mistake.
Thanks in Advance
how do you prepare insert ? i would strongly recommend pcr on digested template and column purification instead gel purification
also this has been discuted before, but you may try column purification rather than gel purification. plasmid is more clean and so you will have more transfromants. At this step, religated plasmid is not the pb as you don't have single colony ! you may dephosphorylate your veector to minimize self religation.